Flt4 (VEGFR-3) as a target for tumor imaging and anti-tumor therapy

ABSTRACT

The present invention provide purified Flt4 receptor tyrosine kinase polypeptides and fragments thereof, polynucleotides encoding such polypeptides, antibodies that specifically bind such polypeptides, and uses therefor.

This application is a continuing application which claims priority fromU.S. patent application Ser. No. 09/169,079 filed Oct. 9, 1998; and fromU.S. patent application Ser. No. 08/901,710, filed Jul. 28, 1997, nowU.S. Pat. No. 6,107,046; and from U.S. patent application Ser. No.08/340,011, filed Nov. 14, 1994, now U.S. Pat. No. 5,776,755; and fromU.S. patent application Ser. No. 08/257,754, filed Jun. 9, 1994, nowabandoned; the latter two of which in turn are continuations-in-part ofU.S. patent application Ser. No. 07/959,951, filed on Oct. 9, 1992, nowabandoned. All of these applications are incorporated herein byreference, in their entireties.

FIELD OF THE INVENTION

The present invention relates generally to genes for receptors,specifically genes for receptor tyrosine kinases, their insertion intorecombinant DNA vectors, and the production of the resulting proteins inhost strains of microorganisms and host eukaryotic cells. Morespecifically, the present invention is directed to Flt4, a receptortyrosine kinase; to nucleotide sequences encoding Flt4; to methods forthe generation of DNAs encoding Flt4 and their gene products; to nucleicacid probes which specifically recognize (hybridize to) nucleic acidsencoding such receptors; to antibodies that specifically recognize suchreceptors; and to methods of using such probes and antibodies and otherFlt4 binding compounds, e.g., for identifying lymphatic vessels and highendothelial venules (HEV) in animal and human tissues and augmenting orpreventing the growth of Flt4-expressing cells in pathologicalconditions.

BACKGROUND

The cellular behavior responsible for the development, maintenance andrepair of differentiated cells and tissues is regulated, in large part,by intercellular signals conveyed via growth factors and similar ligandsand their receptors. The receptors are located on the cell surface ofresponding cells and they bind peptides or polypeptides known as growthfactors as well as other hormone-like ligands. The results of thisinteraction are rapid biochemical changes in the responding cells, aswell as a rapid and a long term readjustment of cellular geneexpression. Several receptors associated with various cell surfaces canbind specific growth factors.

Tyrosine phosphorylation is one of the key modes of signal transductionacross the plasma membrane. Several tyrosine kinase genes encodetransmembrane receptors for polypeptide growth factors and hormones,such as epidermal growth factor (EGF), insulin, insulin-like growthfactor-I (IGF-I), platelet derived growth factors (PDGF-A and -B) andfibroblast growth factors (FGFs) [Heldin et al., Cell Regulation, 1:555-566 (1990); Ullrich et al., Cell, 61: 243-54 (1990)]. The receptorsof several hematopoietic growth factors are tyrosine kinases; theseinclude c-fms, which is the colony stimulating factor 1 receptor [Sherret al., Cell, 41: 665-676 (1985)] and c-kit, a primitive hematopoieticgrowth factor receptor [Huang et al., Cell, 63: 225-33 (1990)].

These receptors differ in their specificity and affinity. In general,receptor tyrosine kinases are glycoproteins, which consist of anextracellular domain capable of binding a specific growth factor(s), atransmembrane domain which is usually an alpha-helical portion of theprotein, a juxtamembrane domain (where the receptor may be regulated by,e.g., protein phosphorylation), a tyrosine kinase domain (which is theenzymatic component of the receptor), and a carboxy-terminal tail, whichin many receptors is involved in recognition and binding of thesubstrates for the tyrosine kinase.

In several receptor tyrosine kinases, the processes of alternativesplicing and alternative polyadenylation are capable of producingseveral distinct polypeptides from the same gene. These may or may notcontain the various domains listed above. As a consequence, someextracellular domains may be expressed as separate proteins secreted bythe cells and some forms of the receptors may lack the tyrosine kinasedomain and contain only the extracellular domain inserted into theplasma membrane via the transmembrane domain plus a shortcarboxy-terminal tail.

The physiology of the vascular system, embryonic vasculogenesis andangiogenesis, blood clotting, wound healing and reproduction, as well asseveral diseases, involve the vascular endothelium lining the bloodvessels. The development of the vascular tree occurs throughangiogenesis, and, according to some theories, the formation of thelymphatic system starts shortly after arterial and venous development bysprouting from veins. See Sabin, F. R., Am. J Anat., 9:43 (1909); andvan der Putte, S. C. J, Adv. Anat. Embryol. Cell Biol., 51:3 (1975).

After the fetal period, endothelial cells proliferate very slowly,except during angiogenesis associated with neovascularization. Growthfactors stimulating angiogenesis exert their effects via specificendothelial cell surface receptor tyrosine kinases.

Among ligands for receptor tyrosine kinases, the Platelet Derived GrowthFactor (PDGF) has been shown to be angiogenic, albeit weakly, in thechick chorioallantoic membrane. Transforming Growth Factor α (TGFα) isan angiogenic factor secreted by several tumor cell types and bymacrophages. Hepatocyte Growth Factor (HGF), the ligand of the c-metproto-oncogene-encoded receptor, is also strongly angiogenic, inducingsimilar responses to those of TGFα in cultured endothelial cells.

Evidence shows that there are endothelial cell specific growth factorsand receptors that may be primarily responsible for the stimulation ofendothelial cell growth, differentiation, as well as certain ofdifferentiated functions. The most-widely studied growth factor isVascular Endothelial Growth Factor (VEGF), a member of the PDGF family.Vascular endothelial growth factor is a dimeric glycoprotein ofdisulfide-linked 23 kDa subunits, discovered because of its mitogenicactivity toward endothelial cells and its ability to induce vesselpermeability (hence its alternative name vascular permeability factor).Other reported effects of VEGF include the mobilization of intracellularCa²⁺, the induction of plasminogen activator and plasminogen activatorinhibitor-1 synthesis, stimulation of hexose transport in endothelialcells, and promotion of monocyte migration in vitro. Four VEGF isoforms,encoded by distinct mRNA splicing variants, appear to be equally capableof stimulating mitogenesis of endothelial cells. The 121 and 165 aminoacid isoforms of VEGF are secreted in a soluble form, whereas theisoforms of 189 and 206 amino acid residues remain associated with thecell surface and have a strong affinity for heparin. Solublenon-heparin-binding and heparin-binding forms have also been describedfor the related placenta growth factor (PIGF; 131 and 152 amino acids,respectively), which is expressed in placenta, trophoblastic tumors, andcultured human endothelial cells.

The pattern of VEGF expression suggests its involvement in thedevelopment and maintenance of the normal vascular system and in tumorangiogenesis. During murine development, the entire 7.5 day post-coitalendoderm expresses VEGF and the ventricular neuroectoderm produces VEGFat the capillary ingrowth stage. On day two of quail development, thevascularized area of the yolk sac as well as the whole embryo showexpression of VEGF. In addition, epithelial cells next to fenestratedendothelia in adult mice show persistent VEGF expression, suggesting arole in the maintenance of this specific endothelial phenotype andfunction.

Two high affinity receptors for VEGF have been characterized,VEGFR-1/Flt1 (fins-like tyrosine kinase-1) and VEGFR-2/Kdr/Flk-1 (kinaseinsert domain containing receptor/fetal liver kinase-1). These receptorsare classified in the PDGF-receptor family. However, the VEGF receptorshave seven immunoglobulin-like loops in their extracellular domains (asopposed to five in other members of the PDGF family) and a longer kinaseinsert. The expression of VEGF receptors occurs mainly in vascularendothelial cells, although some may also be present on monocytes and onmelanoma cell lines. Only endothelial cells have been reported toproliferate in response to VEGF, and endothelial cells from differentsources show different responses. Thus, the signals mediated throughVEGFR-1 and VEGFR-2 appear to be cell type specific.

VEGFR-1 and VEGFR-2 bind VEGF165 with high affinity (K_(d) about 20 pMand 200 pM, respectively). Flk-1 receptor has also been shown to undergoautophosphorylation in response to VEGF, but phosphorylation of Flt1 wasbarely detectable. VEGFR-2 mediated signals cause striking changes inthe morphology, actin reorganization and membrane ruffling of porcineaortic endothelial cells overexpressing this receptor. In these cells,VEGFR-2 also mediated ligand-induced chemotaxis and mitogenicity;whereas VEGFR-1 transfected cells lacked mitogenic responses to VEGF. Incontrast, VEGF had a strong growth stimulatory effect on rat sinusoidalendothelial cells expressing VEGFR-1. Phosphoproteins co-precipitatingwith VEGFR-1 and VEGFR-2 are distinct, suggesting that differentsignalling molecules interact with receptor specific intracellularsequences.

In in situ hybridization studies, mouse VEGFR-2 mRNA expression wasfound in yolk sac and intraembryonic mesoderm (estimated 7.5 daypost-coitum (p.c.) embryos, from which the endothelium is derived, andlater in presumptive angioblasts, endocardium and large and small vesselendothelium (12.5 days p.c.). Abundant VEGFR-2 mRNA in proliferatingendothelial cells of vascular sprouts and branching vessels of embryonicand early postnatal brain and decreased expression in adult brainsuggested that VEGFR-2 is a major regulator of vasculogenesis andangiogenesis. VEGFR-1 expression was similarly associated with earlyvascular development in mouse embryos and with neovascularization inhealing skin wounds. However, high levels of VEGFR-1 expression weredetected in adult organs, suggesting that VEGFR-1 has a function inquiescent endothelium of mature vessels not related to cell growth. Theavian homologue of VEGFR-2 was observed in the mesoderm from the onsetof gastrulation, whereas the VEGFR-1 homologue was first found in cellsco-expressing endothelial markers. In in vitro quail epiblast cultures,FGF-2, which is required for vasculogenic differentiation of thesecells, upregulated VEGFR-2 expression. The expression of both VEGFreceptors was found to become more restricted later in development. Inhuman fetal tissues VEGFR-1 and VEGFR-2 showed overlapping, but slightlydifferent, expression patterns. These data suggest that VEGF and itsreceptors act in a paracrine manner to regulate the differentiation ofendothelial cells and neovascularization of tissues.

VEGF recently has been shown to be a hypoxia-induced stimulator ofendothelial cell growth and angiogenesis, and inhibition of VEGFactivity using specific monoclonal antibodies has been shown to reducethe growth of experimental tumors and their blood vessel density.[Ferrara et al. Endocrine Reviews, 18: 4-25 (1997); Shibuya et al., Adv.Cancer Res., 67: 281-316 (1995); Kim et al., Nature, 362: 841-844(1993).]

Growth of solid tumors beyond a few cubic millimeters in size isdependent on vascular supply, making angiogenesis an attractive targetfor anti-cancer therapy. Encouraging results have been reported withendogenous angiogenic inhibitors or “statins” which include angiostatin,a fragment of plasminogen, and endostatin, a fragment of collagen 18.[O'Reilly et al., Cell, 79: 315-328 (1994); O'Reilly et al., Cell, 88:277-85 (1997).]. Both factors are normally produced by primary tumorsand keep metastasis dormant. Systemic administration of either statinhas been shown to also induce and sustain dormancy of primary tumors inanimal models. The receptors and signalling by statins, as well as theproteases which activate them, remain to be identified. A need existsfor additional therapeutic molecules for controlling angiogenesis in thetreatment of cancer and other pathological disease states.

Primary breast cancers have been shown to express several angiogenicpolypeptides, of which VEGF was the most abundant. [See, e.g., Relf etal., Cancer Res., 57: 963-969 (1997).] Tumor cells contained high levelsof VEGF mRNA in both invasive and non-invasive, ductal (in situ) breastcarcinomas. [Brown et al., Hum. Pathol., 26: 86-91 (1995).] Theendothelial cells adjacent to the in situ carcinomas expressed VEGFR-1and VEGFR-2 mRNA. VEGF and its receptors may contribute to theangiogenic progression of malignant breast tumors, because in severalindependent studies, correlations have been found between tumor vasculardensity and the prognosis of the disease. [Weidner et al., J. Natl.Cancer Inst., 84: 1875-1887 (1992).] A need exists for additionalmarkers for breast cancer and breast cancer-related angiogenesis, toimprove diagnosis and screening and to serve as a target for therapeuticintervention.

A major function of the lymphatic system is to provide fluid return fromtissues and to transport many extravascular substances back to theblood. In addition, during the process of maturation, lymphocytes leavethe blood, migrate through lymphoid organs and other tissues, and enterthe lymphatic vessels, and return to the blood through the thoracicduct. Specialized venules, high endothelial venules (HEVs), bindlymphocytes again and cause their extravasation into tissues. Thelymphatic vessels, and especially the lymph nodes, thus play animportant role in immunology and in the development of metastasis ofdifferent tumors.

Since the beginning of the 20th century, three different theoriesconcerning the embryonic origin of the lymphatic system have beenpresented. However, lymphatic vessels have been difficult to identify,due to the absence of known specific markers available for them.

Lymphatic vessels are most commonly studied with the aid oflymphography. In lymphography, X-ray contrast medium is injecteddirectly into a lymphatic vessel. That contrast medium is distributedalong the efferent drainage vessels of the lymphatic system. Thecontrast medium is collected in lymph nodes, where it stays for up tohalf a year, during which time X-ray analyses allow the follow-up oflymph node size and architecture. This diagnostic is especiallyimportant in cancer patients with metastases in the lymph nodes and inlymphatic malignancies, such as lymphoma. However, improved materialsand methods for imaging lymphatic tissues are needed in the art.

SUMMARY OF THE INVENTION

The present invention addresses a gene for a novel receptor tyrosinekinase located on chromosome 5, identified as an unknown tyrosinekinase-homologous PCR-cDNA fragment from human leukemia cells[Aprelikova et al., Cancer Res., 52: 746-748 (1992)]. This gene and itsencoded protein are called Flt4. This abbreviation comes from the wordsfms-like tyrosine kinase 4.

Flt4 is a receptor tyrosine kinase closely related in structure to theproducts of the VEGFR-1 and VEGFR-2 genes. By virtue of this similarityand subsequently-discovered similarities between ligands for these threereceptors, the Flt4 receptor has additionally been named VEGFR-3. Thenames Flt4 and VEGFR-3 are used interchangeably herein. Despite thesimilarity between these three receptors, the mature form of Flt4differs from the VEGFRs in that it is proteolytically cleaved in theextracellular domain into two disulfide-linked polypeptides of 125/120kD and 75 kD. The Flt4 gene encodes 4.5 and 5.8 kb mRNAs which exhibitalternative 3′ exons and encode polypeptides of 190 kD and 195 kD,respectively.

Further evidence of a distinction is that VEGF does not show specificbinding to Flt4 and doesn't induce its autophosphorylation.

A comparison of Flt4, Flt1, and KDR/Flk-1 receptor mRNA signals showedoverlapping, but distinct expression patterns in the tissues studied.Kaipainen, et al., J. Exp. Med., 178:2077 (1993). Flt4 gene expressionappears to be more restricted than the expression of VEGFR-1 or VEGFR-2.The expression of Flt4 first becomes detectable by in situ hybridizationin the angioblasts of head mesenchyme, the cardinal vein andextraembryonically in the allantois of 8.5 day post-coital mouseembryos. In 12.5 day post-coital embryos the Flt4 signal is observed ondeveloping venous and presumptive lymphatic endothelia, but arterialendothelia appear to be negative. During later stages of development,Flt4 mRNA becomes restricted to developing lymphatic vessels. Only thelymphatic endothelia and some high endothelial venules express Flt4 mRNAin adult human tissues and increased expression occurs in lymphaticsinuses in metastatic lymph nodes and in lymphangioma. The resultssupport the theory of the venous origin of lymphatic vessels.

The protein product of the Flt4 receptor tyrosine kinase cDNA, clonedfrom a human erythroleukemia cell line, is N-glycosylated and containsseven immunoglobulin-like loops in its extracellular domain. Thecytoplasmic tyrosine kinase domain of Flt4 is about 80% identical at theamino acid level with the corresponding domains of Flt1 and KDR andabout 60% identical with the receptors for platelet-derived growthfactor, colony stimulating factor-1, stem cell factor, and the Flt3receptor. See Pajusola et al., Cancer Res., 52:5738 (1992).

The present invention provides isolated polynucleotides (e.g., DNA orRNA segments of defined structure) encoding an Flt4 receptor tyrosinekinase useful in the production of Flt4 protein and peptide fragmentsthereof and in recovery of related genes from other sources.

The present invention provides a recombinant DNA vector containing aheterologous segment encoding the Flt4 receptor tyrosine kinase or arelated protein that is capable of being inserted into a microorganismor eukaryotic cell and that is capable of expressing the encodedprotein.

The present invention provides eukaryotic cells capable of producinguseful quantities of the Flt4 receptor tyrosine kinase and proteins ofsimilar function from many species.

The present invention provides peptides that may be producedsynthetically in a laboratory or by microorganisms, which peptides mimicthe activity of the natural Flt4 receptor tyrosine kinase protein. Inanother embodiment, the invention is directed to peptides which inhibitthe activity of Flt4 receptor tyrosine kinase protein.

Particularly preferred are peptides selected from the group consistingof: (a) a Flt4-short form, the nucleotide and deduced amino acidsequence of which appear in SEQ. ID NOs. 1 and 2; and (b) a second formwith different nucleotide and corresponding amino acid residues at itscarboxyl terminal, i.e., an Flt4-long form, the nucleotide and deducedamino acid sequence of which appear in SEQ. ID NOs. 3 and 4. The Flt4long form has a length of 1363 amino acid residues.

DNA and RNA molecules, recombinant DNA vectors, and modifiedmicroorganisms or eukaryotic cells comprising a nucleotide sequence thatencodes any of the proteins or peptides indicated above are also part ofthe present invention. In particular, sequences comprising all or partof the following two DNA sequences, a complementary DNA or RNA sequence,or a corresponding RNA sequence are especially preferred: (a) a DNAsequence such as SEQ ID NO: 1, encoding Flt4-short form [SEQ ID NO: 2],and (b) a DNA sequence such as SEQ ID NO: 3, encoding a Flt4 whereinnucleotides 3913-4416 of SEQ ID NO: 1 are changed, encoding Flt4-longform [SEQ ID NO: 4].

DNA and RNA molecules containing segments of the larger sequence arealso provided for use in carrying out preferred aspects of the inventionrelating to the production of such peptides by the techniques of geneticengineering and the production of oligonucleotide probes.

Because the DNA sequence encoding the Flt4 protein is identified herein,DNA encoding the Flt4 protein may be produced by, e.g., polymerase chainreaction or by synthetic chemistry using commercially availableequipment, after which the gene may be inserted into any of the manyavailable DNA vectors using known techniques of recombinant DNAtechnology. Furthermore, automated equipment is also available thatmakes direct synthesis of any of the peptides disclosed herein readilyavailable.

The present invention also is directed to Flt4 peptides and otherconstructs and to the use of Flt4 as a specific marker for lymphaticendothelial cells.

In a specific embodiment, the invention is directed to nucleic acidprobes and antibodies recognizing Flt4, especially to monoclonalantibodies, and compositions containing such antibodies.

Also in a specific embodiment, the invention is directed to a method formonitoring lymphatic vessels in tissue samples and in organisms.Further, is it an object of the present invention to provide clinicaldetection methods describing the state of lymphatic tissue, andespecially lymphatic vessels (inflammation, infection, traumas, growth,etc.), and to provide methods for detecting lymphatic vessels, and thuslymphatic vascularization, in an organism.

It is a further object of the present invention to provide monoclonalantibodies which specifically recognize the Flt4 receptor protein orvarious epitopes thereof. It is an object of the invention to use thesemonoclonal antibodies for diagnostic purposes for detecting andmeasuring the amount of Flt4 receptors in tissues, especially inlymphatic tissues. In the context of anti-Flt4 antibodies, the terms“specifically recognize Flt4,” “specifically bind to Flt4,” “specificfor Flt4,” and the like mean that an antibody will bind to (immunoreactwith) Flt4 preferentially over other endothelial cell surface receptors,including VEGFR-2/Kdr/Flk-1 and VEGFR-1/Flt1. Thus, anti-Flt4 antibodiesor other Flt4 binding compounds that are “specific for” Flt4 are usefulfor identification and/or labelling of Flt4 in tissues or biologicalsamples in accordance with the methods of the invention as describedherein (e.g., medical imaging, detection, screening, or targetedtherapy), because they fail to bind epitopes of other antigens at all,or bind other antigens only with an affinity that is sufficiently lowerthan their Flt4 binding affinity to be insignificant in these practicalcontexts.

Another aspect of the present invention relates to a method ofdetermining the presence of Flt4-receptors in a cell sample, comprisingthe steps of: (a) exposing a cell sample to an antibody, especially amonoclonal antibody, of the present invention; and (b) detecting thebinding of said monoclonal antibody to Flt4 receptors. As will beapparent from the detailed description which follows, informationconcerning the presence, quantity, density, and location of Flt4receptor in tissue samples has diagnostic and prognostic relevance withrespect to the type and severity of a disease state; and has therapeuticrelevance where it is desirable to specifically tailor anti-Flt4-basedtreatment regimens to only those patients having diseases characterizedby Flt4 expression in tumors or in blood or lymphatic vessels andtissues surrounding, serving, or supplying a tumor. The screening forthe presence of Flt4 receptors thus can constitute a first step in atherapeutic regimen, and/or a monitoring step during the course oftherapy.

The invention is further directed to a method of modulating (e.g.,antagonizing or augmenting) the function of Flt4 in lymphaticvascularization and in inflammatory, infectious and immunologicalconditions. For example, in one embodiment, such a method comprisesinhibiting the Flt4-mediated lymphatic vascularization by providingamounts of a Flt4-binding compound sufficient to block the Flt4endothelial cell sites participating in such reaction, especially whereFlt4 function is associated with a disease such as metastatic cancers,lymphomas, inflammation (chronic or acute), infections and immunologicaldiseases. Since many tumors metastasize through the lymphatic vessels,therapy directed to blocking the interaction between Flt4 ligands andFlt4 is expected to have broad application for inhibition of tumormetastasis as part of an anti-cancer treatment regimen.

The invention is further directed to a specific Flt4-stimulating ligandand monoclonal antibodies and their use for stimulating lymphaticendothelia and fragments and peptides as well as antibodies derived fromresearch on the ligand to inhibit Flt4 function when desirable, such asin various disease states involving Flt4 function.

The invention provides a cell line source for the ligand of the Flt4receptor tyrosine kinase. Using the conditioned medium from these cells,the Flt4 ligand may be purified and cloned by using methods standard inthe art. Using this conditioned medium or the purified ligand, an assaysystem for Flt4 ligand and dimerization inhibitors as well as inhibitorsof Flt4 signal transduction are obtained, which allow for identificationand preparation of such inhibitors.

In a preferred embodiment of the invention, conditioned medium from thePC-3 cell line comprises a protein or a fragment thereof, which iscapable of stimulating the Flt4 receptor and regulating the growth anddifferentiation as well as the differentiated functions of certainendothelial cells. The Flt4 ligand or its peptides or derivatives areuseful in the regulation of endothelial cell growth, differentiation andtheir differentiated functions and in the generation of agonists andantagonists for the ligand. Particularly, the Flt4 ligand is useful inregulating lymphatic endothelia. However, the Flt4 ligand, whenpurified, or produced from a recombinant source, may also stimulate therelated KDR/Flk-1 receptor.

The identification of Flt4-stimulating ligand makes it directly possibleto assay for inhibitors of this ligand or inhibitors of Flt4 function.Such inhibitors are simply added to the conditioned media containing theFlt4 ligand and if they inhibit autophosphorylation, they act as Flt4signalling inhibitors. For example, recombinant or synthetic peptides(including but not limited to fragments of the Flt4 extracellulardomain) may be assayed for inhibition of Flt4-ligand interaction or Flt4dimerization. Such putative inhibitors of Flt4 and, in addition,antibodies against the Flt4 ligand, peptides or other compounds blockingFlt4 receptor-ligand interaction, as well as antisense oligonucleotidescomplementary to the sequence of mRNA encoding the Flt4 ligand areuseful in the regulation of endothelial cells and in the treatment ofdiseases associated with endothelial cell function.

A detailed characterization of the Flt4 ligand, designated VEGF-C, isprovided in PCT Patent Application No. PCT/US98/01973, filed 2 Feb.1998, and published as International Publication No. WO 98/33917; in PCTPatent Application PCT/FI96/00427, filed Aug. 1, 1996, and published asInternational Publication WO 97/05250; and in the U.S. PatentApplication priority documents relied upon therein for priority, all ofwhich are incorporated herein by reference. The deduced amino acidsequence for prepro-VEGF-C is set forth herein in SEQ ID NO: 21.

A detailed description of a second Flt4 ligand, designated VEGF-D, isprovided in Achen, et al., Proc. Nat'l Acad. Sci. U.S.A., 95(2): 548-553(1998), and in Genbank Accession No. AJ000185, both of which areincorporated herein by reference. The deduced amino acid sequence forprepro-VEGF-D is set forth herein in SEQ ID NO: 22.

The invention also is directed to a method of treating a mammalianorganism suffering from a disease characterized by expression of Flt4tyrosine kinase (Flt4) in cells, comprising the step of administering tothe mammalian organism a composition, the composition comprising acompound effective to inhibit the binding of an Flt4 ligand protein toFlt4 expressed in cells of the organism, thereby inhibiting Flt4function. The disease may be diseases already mentioned above, such asdiseases characterized by undesirable lymphatic vascularization.Additionally, it has been discovered that Flt4 expression also occurs inblood vessel vasculature associated with at least some breast cancers,and possibly other cancers (i.e., at a level greatly exceeding thebarely detectable or undetectable levels of expression in blood vesselvasculature of corresponding normal (healthy) tissue). Thus, in apreferred embodiment, the cells comprise endothelial cells (lymphatic orvascular). In another embodiment, the cells comprise neoplastic cellssuch as certain lymphomas that express Flt4. Treatment of humans isspecifically contemplated.

By “compound effective to inhibit the binding of an Flt4 ligand proteinto Flt4 expressed in cells of the organism” is meant any compound thatinhibits the binding of the Flt4 ligand described herein as vascularendothelial growth factor C, as isolatable from PC-3 conditioned medium.It is contemplated that such compounds also will be effective forinhibiting the binding of vascular endothelial growth factor D to Flt4.Exemplary compounds include the following polypeptides: (a) apolypeptide comprising an antigen-binding fragment of an anti-Flt4antibody; (b) a polypeptide comprising a soluble Flt4 fragment (e.g., anextracellular domain fragment), wherein the fragment and the polypeptideare capable of binding to an Flt4 ligand; (c) a polypeptide comprising afragment or analog of a vertebrate vascular endothelial growth factor C(VEGF-C) polypeptide, wherein the polypeptide and the fragment or analogbind, but fail to activate, the Flt4 expressed on native host cells(i.e., cells of the organism that express the native Flt4 protein ontheir surface); and (d) a polypeptide comprising a fragment or analog ofa vertebrate vascular endothelial growth factor-D (VEGF-D) polypeptide,wherein the polypeptide and the fragment or analog bind, but fail toactivate, the Flt4 expressed on native host cells. Small moleculeinhibitors identifiable by standard in vitro screening assays, e.g.,using VEGF-C and recombinantly-expressed Flt4 also are contemplated.Polypeptides comprising an antigen-binding fragment of an anti-Flt4antibody are highly preferred. Such polypeptides include, e.g.,polyclonal and monoclonal antibodies that specifically bind Flt4;fragments of such antibodies; chimaeric and humanized antibodies;bispecific antibodies that specifically bind to Flt4 and alsospecifically bind to another antigen, and the like. Use of compoundsthat bind to circulating Flt4 ligand and thereby inhibit the binding ofthe ligand to Flt4 also is contemplated. Such compounds includeanti-VEGF-C or anti-VEGF-D antibodies or polypeptides that compriseantigen-binding fragments thereof. In a related variation, the inventioncontemplates methods of treatment that disrupt downstream intracellularFlt4 signalling, thereby inhibiting Flt4 function.

In a preferred variation, the compound further comprises a detectablelabel as described elsewhere herein, or a cytotoxic agent. Exemplarycytotoxic agents include plant toxins (e.g., ricin, saporin), bacterialor fungal toxins, radioisotopes (e.g., 211-Astatine, 212-Bismuth,90-Yttrium, 131-Iodine, 99m-Technitium, and others described herein),anti-metabolite drugs (e.g., methotrexate, 5-fluorodeoxyuridine),alkylating agents (e.g., chlorambucil), anti-mitotic agents (e.g., vincaalkaloids), and DNA intercalating agents (e.g., adriamycin). Otherexemplary agents include compounds or treatments that induce DNA damagewhen applied to a cell. Such agents and factors include radiation andwaves that induce DNA damage such as, gamma-irradiation, X-rays,UV-irradiation, microwaves, electronic emissions, and the like. Avariety of chemical compounds, also described as “chemotherapeuticagents,” function to induce DNA damage, all of which are intended to beof use in the combined treatment methods disclosed herein.Chemotherapeutic agents contemplated to be of use, include, e.g.,adriamycin, 5-fluorouracil (5FU), etoposide (VP-16), camptothecin,actinomycin-D, mitomycin C, cisplatin (CDDP) and even hydrogen peroxide.The invention also encompasses the use of a combination of one or moreDNA damaging agents, whether radiation-based or actual compounds, suchas the use of X-rays with cisplatin or the use of cisplatin withetoposide. Still other agents are adriamycin (also known asdoxorubicin), VP-16 (also known as etoposide), and the like,daunorubicin (intercalates into DNA, blocks DNA-directed RNA polymeraseand inhibits DNA synthesis); mitomycin (also known as mutamycin and/ormitomycin-C); Actinomycin D; vincristine and cyclophosphamide;Bleomycin; VP 16 (etoposide); Tumor Necrosis Factor [TNF;] Taxol;Melphalan; Cyclophosphamide, Chlorambucil. Administration of thepeptides of the present invention may precede or follow the other agenttreatment by intervals ranging from minutes to weeks. In embodimentswhere the other agent and expression construct are administeredseparately, one would generally ensure that a significant period of timedid not expire between the time of each delivery, such that the agentand the peptide-based therapeutic would still be able to exert anadvantageously combined effect. In such instances, it is contemplatedthat one would administer both modalities within about 12-24 hours ofeach other and, more preferably, within about 6-12 hours of each other,with a delay time of only about 12 hours being most preferred. In somesituations, it may be desirable to extend the time period for treatmentsignificantly, however, where several days (2, 3, 4, 5, 6 or 7) toseveral weeks (1, 2, 3, 4, 5, 6, 7 or 8) lapse between the respectiveadministrations. Repeated treatments with one or both agents isspecifically contemplated.

Likewise, to improve administration, the composition preferably furthercomprises a pharmaceutically acceptable diluent, adjuvant, or carriermedium.

As explained in detail herein, Flt4 expression, while largely restrictedto the lymphatic endothelia of healthy adults, has been identified inthe blood vasculature surrounding at least certain tumors. Thus, theinvention further includes a method of treating a mammalian organismsuffering from a neoplastic disease characterized by expression of Flt4tyrosine kinase (Flt4) in vascular endothelial cells, comprising thesteps of: administering to a mammalian organism in need of suchtreatment a composition, the composition comprising a compound effectiveto inhibit the binding of an Flt4 ligand protein to Flt4 expressed invascular endothelial cells of the organism, thereby inhibitingFlt4-mediated proliferation of the vascular endothelial cells. Treatmentof neoplastic diseases selected from carcinomas (e.g., breastcarcinomas), squamous cell carcinomas, lymphomas, melanomas, andsarcomas, are specifically contemplated. However, it will be readilyapparent that the screening techniques described herein in detail willidentify other tumors characterized by Flt4 expression in vascularendothelial cells, which tumors are candidates susceptible to theanti-Flt4 treatment regimens described herein. Treatment of breastcarcinomas characterized by expression of Flt4 in vascular endothelialcells is specifically contemplated. By neoplastic disease characterizedby expression of Flt4 tyrosine kinase in vascular endothelial cells ismeant a disease wherein Flt4 is identifiable in blood vasculature at amuch higher level than the undetectable or barely detectable levelsnormally observed in the blood vascular of healthy tissue, asexemplified herein.

Therapeutically effective amounts of compounds are empiricallydetermined using art-recognized dose-escalation and dose-responseassays. By therapeutically effective for treatment of tumors is meant anamount effective to reduce tumor growth, or an amount effective to stoptumor growth, or an amount effective to shrink or eliminate tumorsaltogether, without unacceptable levels of side effects for patientsundergoing cancer therapy. Where the compound comprises an antibody orother polypeptide, doses on the order of 0.1 to 100 mg antibody perkilogram body weight, and more preferably 1 to 10 mg/kg, arespecifically contemplated. For humanized antibodies, which typicallyexhibit a long circulating half-life, dosing at intervals ranging fromdaily to every other month, and more preferably every week, or everyother week, or every third week, are specifically contemplated.Monitoring the progression of the therapy, patient side effects, andcirculating antibody levels will provide additional guidance for anoptimal dosing regimen. Data from published and ongoing clinical trialsfor other antibody-based cancer therapeutics (e.g., anti-HER2, anti-EGFreceptor) also provide useful dosing regimen guidance.

For therapeutic methods described herein, preferred compounds includepolypeptides comprising an antigen-binding fragment of an anti-Flt4antibody, and polypeptides comprising a soluble Flt4 extracellulardomain fragment. Human and humanized anti-Flt4 antibodies are highlypreferred. Highly preferred Flt4 extracellular domain fragments compriseligand binding portions of the Flt4 extracellular domain. For example, asoluble fragment comprising the first three immunoglobulin-like domainsof the Flt4 extracellular domain is highly preferred. Smaller fragmentsthat bind Flt4 ligands alone, or when fused to other peptides (such asimmunoglobulin-like domains of VEGFR-1 or VEGFR-2), also arecontemplated. Similarly, modifications which improve solubility and/orstability, serum half-life, or other properties to improve therapeuticefficacy are contemplated. For example, polypeptides comprising fusionsbetween Flt4 extracellular domain and an immunoglobulin Fc peptide(especially an IgG1 Fc isotype) to improve solubility and serum-halflife, are contemplated. [Compare Achen et al., Proc. Natl. Acad. Sci.USA, 95: 548-553 (1998).]

An expected advantage of the therapeutic methods of the invention liesin the fact that Flt4 is normally not expressed at any significant levelin the blood vasculature of healthy tissues. In a highly preferredembodiment, the therapeutic compound comprises a bispecific antibody, orfragment thereof, wherein the antibody or fragment specifically bindsFlt4 and specifically binds a blood vascular endothelial marker antigen.By “blood vascular endothelial marker antigen” is meant any cell surfaceantigen that is expressed on proliferating vascular endothelial cells,and, preferably, that is not expressed on lymphatic endothelial cells.Exemplary blood vascular endothelial markers include PAL-E [deWaal, etal. Am. J. Pathol., 150:1951-1957 (1994)], VEGFR-1 and VEGFR-2 [Ferraraet al., Endocrine Reviews, 18:4-25 (1997], Tie [Partanen et al., Mol.Cell. Biol., 12: 1698-1707 (1992)], endoglin [U.S. Pat. No. 5,776,427,incorporated herein by reference in its entirety], and von WillebrandtFactor. Such bispecific antibodies are expected to preferentially locateto the tumor-associated vasculature that expresses both Flt4 and theblood vascular endothelial marker. In a highly preferred embodiment, thecompound further comprises an anti-neoplastic or cytotoxic agentconjugated to the bispecific antibody, for the purposes of killing thetumor cells and/or killing the vasculature supply to the tumor cells.Exemplary agents include those described above, and also therapeuticproteins, such as statins, cytokines, chemokines, and the like, tostimulate an immune response to the tumor in the host.

In an alternative embodiment, the compound comprises an antibody (orbispecific antibody) that recognizes an epitope (or epitopes) comprisedof an Flt4/Flt4 ligand complex (e.g., a complex comprised of Flt4 boundto VEGF-C or VEGF-D).

It is further contemplated that the therapeutic compound will beconjugated or co-administered with broad spectrum agents that havepotential to inhibit angiogenic factors. Such agents include, e.g.,heparin binding drugs (such as pentosan and suramin analogs) that mayinhibit angiogenic factors that bind heparin; and chemical agents thatblock endothelial cell growth and migration, such as fumagillin analogs.Other agents currently under investigation include Marimastat (BritishBiotech, Annapolis Md.; indicated for non-small cell lung, small celllung and breast cancers); AG3340 (Agouron, LaJolla, Calif.; forglioblastoma multiforme); COL-3 (Collagenex, Newtown Pa.; for braintumors); Neovastat (Aeterna, Quebec, Canada; for kidney and non-smallcell lung cancer) BMS-275291 (Bristol-Myers Squibb, Wallingford Conn.;for metastatic non-small cell ling cancer); Thalidomide (Celgen; formelanoma, head and neck cancer, ovarian, metastatic prostate, andKaposi's sarcoma; recurrent or metastatic colorectal cancer (withadjuvants); gynecologic sarcomas, liver cancer; multiple myeloma; CLL,recurrent or progressive brain cancer, multiple myeloma, non-small celllung, nonmetastatic prostate, refractory multiple myeloma, and renalcancer); Squalamine (Magainin Pharmaceuticals Plymouth Meeting, Pa.;non-small cell cancer and ovarian cancer); Endostatin (EntreMEd,Rockville, Md.; for solid tumors); SU5416 (Sugen, San Francisco, Calif.;recurrent head and neck, advanced solid tumors, stage IIIB or IV breastcancer; recurrent or progressive brain (pediatric); Ovarian, AML;glioma, advanced malignancies, advanced colorectal, von-Hippel Lindaudisease, advanced soft tissue; prostate cancer, colorectal cancer,metastatic melanoma, multiple myeloma, malignant mesothelioma:metastatic renal, advanced or recurrent head and neck, metastaticcolorectal cancer); SU6668 (Sugen San Francisco, Calif.; advancedtumors); interferon-α; Anti-VEGF antibody (NAtional Cancer Institute,Bethesda Md.; Genentech San Franscisco, Calif.; refractory solid tumors;metastatic renal cell cancer, in untreated advanced colorectal);EMD121974 (Merck KCgaA, Darmstadt, Germany; HIV related Kaposi'sSarcoma, progressive or recurrent Anaplastic Glioma); Interleukin 12(Genetics Institute, Cambridge, Mass.; Kaposi's sarcoma) and IM862(Cytran, Kirkland, Wash.; ovarian cancer, untreated metastatic cancersof colon and rectal origin and Kaposi's sarcoma).

Conjugation of the anti-Flt4 compound to a prodrug that would betargeted to tumor vessels by the anti-Flt4 compound and then activated(e.g., by irradiation) locally at sites of tumor growth also iscontemplated. Use of such prodrug strategy has the expected advantage ofminimizing side effects of the drug upon healthy lymphatic vessels thatexpress Flt4.

Similarly, the invention includes a method of treating a mammalianorganism suffering from a neoplastic disease characterized by expressionof Flt4 tyrosine kinase (Flt4) in vascular endothelial cells, comprisingthe steps of: identifying a mammalian organism suffering from aneoplastic disease state characterized by expression of Flt4 in vascularendothelial cells, and administering to the mammalian organism in needof such treatment a composition, the composition comprising a compoundeffective to inhibit the binding of an Flt4 ligand protein to Flt4expressed in vascular endothelial cells of the organism, therebyinhibiting Flt4-mediated proliferation of the vascular endothelialcells.

The invention also provides a method for screening a biological samplefor the presence of Flt4 receptor tyrosine kinase protein (Flt4),comprising the steps of: (a) contacting a biological sample suspected ofcontaining Flt4 with a composition comprising an Flt4 binding compound,under conditions wherein the compound will bind to Flt4 in thebiological sample; (b) washing the biological sample under conditionsthat will remove Flt4 binding compound that is not bound to Flt4 in thesample; and (c) screening the sample for the presence of Flt4 bydetecting Flt4 binding compound bound to Flt4 receptor tyrosine kinasein the sample after the washing step. Preferably, the compound comprisesa polypeptide selected from the group consisting of: (a) a polypeptidecomprising an antigen-binding fragment of an anti-Flt4 antibody; and (b)a polypeptide comprising an Flt4 ligand or Flt4 binding fragment oranalog thereof. Antibodies that specifically bind Flt4, and that furthercomprise a detectable label, are highly preferred.

The invention also is directed to a method for imaging vertebrate tissuesuspected of containing cells that express Flt4 receptor tyrosine kinaseprotein (Flt4), comprising the steps of: (a) contacting vertebratetissue with a composition comprising an Flt4 binding compound; and (b)imaging the tissue by detecting the Flt4 binding compound bound to thetissue. Preferably, the tissue is human tissue, and the method furthercomprises the step of washing the tissue, after the contacting step andbefore the imaging step, under conditions that remove from the tissueFlt4 compound that is not bound to Flt4 in the tissue.

In a related variation, the invention provides a method for imagingtumors in tissue from a vertebrate organism, comprising the steps of:(a) contacting vertebrate tissue suspected of containing a tumor with acomposition comprising an Flt4 binding compound; (b) detecting the Flt4binding compound bound to cells in said tissue; and (c) imaging solidtumors by identifying blood vessel endothelial cells bound by the Flt4binding compound, wherein blood vessels expressing Flt4 are correlatedwith the presence and location of a tumor in the tissue. In onepreferred embodiment, the method further comprises steps of contactingthe tissue with a second compound (such as an antibody) thatspecifically binds to a blood vessel endothelial marker (e.g., PAL-E,VEGFR-1, VEGFR-2) that is substantially absent in lymphatic endothelia;and detecting the second compound bound to cells in the tissue; whereinthe imaging step comprises identifying blood vessels labeled with boththe Flt4 binding compound and the second compound, and wherein bloodvessels labeled with both the Flt4 binding compound and the secondcompound correlate with the presence and location of a tumor in thetissue. It will be appreciated that the use of the second compound helpsthe practitioner to more rapidly distinguish between blood vessels thatare expressing Flt4 and normal lymphatic vessels which express Flt4 ontheir surface.

The invention is further directed to a method of screening for aneoplastic disease state, comprising the steps of: (a) contacting tissuefrom a mammalian organism suspected of having a neoplastic disease statewith a composition comprising an antibody or antibody fragment thatspecifically binds Flt4 receptor tyrosine kinase; (b) detecting theantibody or antibody fragment bound to cells in the mammalian organism;and (c) screening for a neoplastic disease from the quantity ordistribution of the antibody bound to cells in the mammalian organism.As described herein, Flt4 (which usually is undetectable or barelydetectable in the blood vasculature) is strongly stained in the bloodvasculature of at least some tumors. Thus, in one embodiment, in thescreening step, the detection of the antibody or antibody fragment boundto blood vessel endothelial cells is correlated with the presence of aneoplastic disease. In this method, it will be understood that“detection” means detection at a level significantly higher than thebarely detectable or undetectable levels that would occur incorresponding normal (healthy) tissue, as described herein. Suchdifferential expression can be confirmed by comparison to a controlperformed with tissue from a healthy organism. Screening mammary tissuefor neoplasms is specifically contemplated. As described above, thepractice of such methods may be further facilitated by the administeringto said mammal of a second compound that specifically binds to a bloodvessel endothelial marker, wherein the detecting step comprisesdetection of said first and said second compound bound to neovascularendothelial cells.

From the foregoing it will further be appreciated that the variouscompounds described for use in methods of the invention also areintended as aspects of the invention. Such compounds include theanti-Flt4 antibodies and bi-specific antibodies described above, forexample. Likewise, the use of any compounds described herein (alone orin combination) for the manufacture of a medicament for therapeutic ordiagnostic or imaging purposes described herein also is intended as anaspect of the invention. The medicament may further comprisepharmaceutically acceptable diluents, adjuvants, carriers, or the like.

Similarly, the invention includes kits which comprise compounds orcompositions of the invention packaged in a manner which facilitatesthere use to practice methods of the invention. In a simplestembodiment, such a kit includes a compound or composition of theinvention packaged in a container such as a sealed bottle or vessel,with a label affixed to the container or included in the package thatdescribes use of the compound or composition to practice the method ofthe invention. Preferably, the compound or composition is packaged in aunit dosage form. In another embodiment, a kit of the invention includesa Flt4 binding compound packaged together with a second compound thatbinds to a marker (antigen) that is expressed on the surface of bloodvessel endothelial cells but is substantially absent from lymphaticendothelia.

Additionally, many aspects of the invention have been described in thecontext of using peptides or polypeptides for imaging or therapy, and/orfor using Flt4 protein expression on cell surfaces as a target fordetection, screening, imaging, or the like, using antibodies. Thetherapeutic delivery of protein therapeutics, such as polypeptidescomprising ligand-binding soluble Flt4 fragments, can also beaccomplished with gene therapy materials and methods. For example, anaked DNA construct or gene therapy expression vector constructcomprising a polynucleotide encoding the therapeutic peptide of interestis delivered to a mammalian subject in need of therapy. Preferably, theconstruct comprises a promoter or other expression control sequenceoperatively linked to the sequence encoding the therapeutic peptide, topromote expression of the therapeutic peptide in vivo. In one variation,the nucleic acid is encapsulated in a liposome. In another variation,the nucleic acid is a viral vector such as a retrovirus, adenovirus,adeno-associated virus, vaccinia virus, herpesvirus, or other vectordeveloped for gene therapy protocols in mammals. Exemplary treatmentmethods include steps of administering a pharmaceutical compositioncomprising the gene therapy construct to a patient, or a step oftransforming or transfecting cells ex vivo and introducing thetransformed cells into the patient. Similarly, the detection of Flt4expression by cells or tissues can be performed using polynucleotideprobes that will specifically hybridize to Flt4 mRNA sequences inNorthern hybridization or in situ hybridization assays; or by performingquantitative PCR or other techniques to measure Flt4 mRNA in samples.

Additional features and variations of the invention will be apparent tothose skilled in the art from the entirety of this application,including the detailed description, and all such features are intendedas aspects of the invention. Likewise, features of the inventiondescribed herein can be re-combined into additional embodiments thatalso are intended as aspects of the invention, irrespective of whetherthe combination of features is specifically mentioned above as an aspector embodiment of the invention. Also, only such limitations which aredescribed herein as critical to the invention should be viewed as such;variations of the invention lacking limitations which have not beendescribed herein as critical are intended as aspects of the invention.

In addition to the foregoing, the invention includes, as an additionalaspect, all embodiments of the invention narrower in scope in any waythan the variations specifically mentioned above. Although theapplicant(s) invented the full scope of the claims appended hereto, theclaims appended hereto are not intended to encompass within their scopethe prior art work of others. Therefore, in the event that statutoryprior art within the scope of a claim is brought to the attention of theapplicants by a Patent Office or other entity or individual, theapplicant(s) reserve the right to exercise amendment rights underapplicable patent laws to redefine the subject matter of such a claim tospecifically exclude such statutory prior art or obvious variations ofstatutory prior art from the scope of such a claim. Variations of theinvention defined by such amended claims also are intended as aspects ofthe invention.

BRIEF DESCRIPTION OF THE-DRAWINGS

FIG. 1A is a schematic depiction of the structure of Flt4 cDNA clones;

FIG. 1B is a photographic reproduction of a Northern hybridization gel;

FIGS. 2A-F present a schematic depiction of structural features of Flt4and a comparison with the Flt1 tyrosine kinase sequence;

FIG. 3A is a schematic depiction of the 3′ ends of the cDNA inserts ofclones J.1.1 and I.1.1;

FIG. 3B is a photographic reproduction of autoradiograms ofhybridizations with anti-sense RNA probe and the long and short forms ofFlt4 RNA;

FIG. 3C is a photographic reproduction of autoradiograms ofhybridizations with anti-sense RNA probe and the long and short forms ofFlt4 RNA;

FIG. 4 is a photographic reproduction of a gel illustrating ahybridization analysis of Flt4 sequences in DNA samples from differentspecies;

FIGS. 5A-5H depict immunohistochemical characterization ofVEGFR-3-expressing vessels in intraductal carcinoma. In adjacentsections (FIGS. 5A, B), VEGFR-3 and PAL-E decorate a similar pattern of“necklace” vessels (arrowheads) around the duct filled with carcinomacells. Another set of adjacent sections was compared with staining forVEGFR-3 (FIG. 5C), laminin (FIG. 5D), collagen XVIII (FIG. 5E) and SMA(FIG. 5F). Double staining for PAL-E and VEGFR-3 (FIG. 5G) andcomparison with adjacent section stained for VEGFR-3 only (FIG. 5H). Thevessels adjacent to the affected ducts are double-positive (arrowheads),whereas a VEGFR-3 positive vessel is present a short distance away fromthe affected duct in the interductal stroma (arrows). Note that basallamina is positive for PAL-E in the double staining procedure.Magnifications: FIGS. 5A,B 400 x. FIGS. 5C, D, E, F 320×. FIGS. 5E,F480×.

DETAILED DESCRIPTION

The cloning, sequencing and expression of a novel receptor tyrosinekinase, termed Flt4, is described below. The Flt4 gene maps tochromosomal region 5q35 where many growth factors and growth factorreceptors are located. The extracellular domain of Flt4 consists ofseven immunoglobulin-like loops including twelve potential glycosylationsites. On the basis of structural similarities, Flt4 and the previouslyknown Flt1 and KDR/FLK1 receptors may constitute a subfamily of classIII tyrosine kinases. The Flt4 gene is expressed as 5.8 kb and 4.5 kbmRNAs which were found to differ in their 3′ sequences and to bedifferentially expressed in HEL and DAMI leukemia cells.

A Wilm's tumor cell line, a retinoblastoma cell line, and anondifferentiated teratocarcinoma cell line expressed Flt4; whereasdifferentiated teratocarcinoma cells were negative. Most fetal tissuesalso expressed the Flt4 mRNA, with spleen, brain intermediate zone andlung showing the highest levels. In human adult tissues the highestexpression level was found in placenta, lung, kidney, heart and liver indecreasing order of expression. In in situ hybridization, the Flt4autoradiographic grains decorated endothelial cells of fetal lung.Immunohistochemical staining of Flt4 in fetal tissues confirmed stainingof the endothelial cells. The expression pattern of Flt4 in comparisonto Flt1 and KDR differs greatly in tissues of 18-week-old human fetuses.See Kaipainen et al., J. Exp. Med., 178:2077 (1993).

Expression vectors containing the Flt4 cDNA have been produced andexpressed in COS and NIH3T3 cells as described in Examples 4 and 11.

The Flt4 DNAs and polypeptides of the invention may be useful in thepurification of the Flt4 ligand, and in the regulation of growth anddifferentiation of endothelial cells in various organs. They may alsoprove valuable in the diagnosis/treatment of certain diseases.

In the description that follows, a number of terms used in recombinantDNA (rDNA) technology are extensively utilized. In order to provide aclear and consistent understanding of the specification and claims,including the scope to be given to such terms, the following definitionsare provided.

Gene. A DNA sequence containing a template for a RNA polymerase. The RNAtranscribed from a gene may or may not code for a protein. RNA thatcodes for a protein is termed messenger RNA (mRNA) and, in eukaryotes,is transcribed by RNA polymerase II. However, it is also known toconstruct a gene containing a RNA polymerase II template wherein a RNAsequence is transcribed which has a sequence complementary to that of aspecific mRNA but is not normally translated. Such a gene construct isherein termed an “antisense RNA gene” and such a RNA transcript istermed an “antisense RNA.” Antisense RNAs' are not normally translatabledue to the presence of translational stop codons in the antisense RNAsequence.

A “complementary DNA” or “cDNA” gene includes recombinant genessynthesized by reverse transcription of mRNA lacking interveningsequences (introns).

Cloning vehicle. A plasmid or phage DNA or other DNA sequence which isable to replicate autonomously in a host cell, and which ischaracterized by one or a small number of endonuclease recognition sitesat which such DNA sequences may be cut in a determinable fashion withoutloss of an essential biological function of the vehicle, and into whichDNA may be spliced in order to bring about its replication and cloning.The cloning vehicle may further contain a marker suitable for use in theidentification of cells transformed with the cloning vehicle. Markers,for example, are tetracycline resistance or ampicillin resistance. Theword “vector” is sometimes used for “cloning vehicle.”

Expression vector. A vehicle or vector similar to a cloning vehicle andwhich is capable of expressing a gene which has been cloned into it,after transformation into a host. The cloned gene is usually placedunder the control of (i.e., operably linked to) certain controlsequences such as promoter sequences. Expression control sequences varydepending on whether the vector is designed to express the operablylinked gene in a prokaryotic or eukaryotic host and may additionallycontain transcriptional elements such as enhancer elements, terminationsequences, tissue-specificity elements, and/or translational initiationand termination sites.

The present invention pertains to both expression of recombinant Flt4proteins (short and long forms), and to the functional derivatives ofthese proteins.

Functional Derivative. A “functional derivative” of Flt4 proteins is aprotein which possesses a biological activity (either functional orstructural) that is substantially similar to a biological activity ofnon-recombinant Flt4 proteins. A functional derivative of the Flt4protein may or may not contain post-translational modifications such ascovalently linked carbohydrate, depending on the necessity of suchmodifications for the performance of a specific function. The term“functional derivative” is intended to include the “fragments,”“variants,” “analogues,” and “chemical derivatives” of a molecule.

As used herein, a molecule is said to be a “chemical derivative” ofanother molecule when it contains additional chemical moieties notnormally a part of the molecule. Such moieties may improve themolecule's solubility, absorption, biological half-life, etc. Themoieties may alternatively decrease the toxicity of the molecule andeliminate or attenuate any undesirable side effect of the molecule, etc.Moieties capable of mediating such effects are disclosed in Remington'sPharmaceutical Sciences (1980). Procedure for coupling such moieties toa molecule are well known in the art.

Fragment. A “fragment” of a molecule such as Flt4 protein is meant torefer to any portion of the molecule, such as the peptide core, or avariant of the peptide core.

Variant. A “variant” of a molecule such as Flt4 protein is meant torefer to a molecule substantially similar in structure and biologicalactivity to either the entire molecule, or to a fragment thereof. Thus,provided that two molecules possess a similar activity, they areconsidered variants as that term is used herein even if the compositionor secondary, tertiary, or quaternary structure of one of the moleculesis not identical to that found in the other, or if the sequence of aminoacid residues is not identical.

Analogue. An “analogue” of Flt4 protein or genetic sequence is meant torefer to a protein or genetic sequence substantially similar in functionto the Flt4 protein or genetic sequence herein.

DESCRIPTION OF PREFERRED EMBODIMENTS

The present invention is directed to what applicants have termed “Flt4,”a receptor for tyrosine kinase, Flt4-encoding nucleic acid molecules(e.g. cDNAs, genomic DNAs, RNAs, anti-sense RNAs, etc.), production ofFlt4 peptides or Flt4 protein from a Flt4 gene sequence and its product,recombinant Flt4 expression vectors, Flt4 analogues and derivatives, anddiagnostic and/or therapeutic uses of Flt4 and related proteins, Flt4ligands, Flt4 antagonists and anti-Flt4 antibodies.

Production of Recombinant Flt4

Biologically active Flt4 may be produced by the cloning and expressionof the Flt4-encoding sequence or its functional equivalent in a suitablehost cell.

Production of Flt4 using recombinant DNA technology may be divided intoa step-wise process for the purpose of description: (1) isolating orgenerating the coding sequence (gene) for the desired Flt4; (2)constructing an expression vector capable of directing the synthesis ofthe desired Flt4; (3) transfecting or transforming appropriate hostcells capable of replicating and expressing the Flt4 gene and/orprocessing the gene product to produce the desired Flt4; and (4)identifying and purifying the desired Flt4 product.

Isolation or Generation of the Flt4 Gene

The nucleotide coding sequence of Flt4 or functional equivalentsthereof, may be used to construct recombinant expression vectors whichwill direct the expression of the desired Flt4 product. In the practiceof the method of the invention, the nucleotide sequence depictedtherein, or fragments or functional equivalents thereof, may be used togenerate the recombinant molecules which will direct the expression ofthe recombinant Flt4 product in appropriate host cells. Flt4-encodingnucleotide sequences may be obtained from a variety of cell sourceswhich produce Flt4-like activities and/or which express Flt4-encodingmRNA. Applicants have identified a number of suitable human cell sourcesfor Flt4, including human placenta, leukemia cells and some tumor celllines.

The Flt4 coding sequence may be obtained by cDNA cloning from RNAisolated and purified from such cell sources or by genomic cloning. TheFlt4 sequence may be for example amplified by polymerase chain reactionfrom cDNA or genomic DNA material using techniques well known in theart. Either cDNA or genomic libraries of clones may be prepared usingtechniques well known in the art and may be screened for particular Flt4DNAs with nucleotide probes which are substantially complementary to anyportion of the Flt4 gene. Full length clones, i.e., those containing theentire coding region of the desired Flt4, may be selected forconstructing expression vectors. Alternatively, Flt4 encoding DNAs maybe synthesized in whole or in part by chemical synthesis usingtechniques standard in the art. Due to the inherent degeneracy ofnucleotide coding sequences, other DNA sequences which encodesubstantially the same or a functionally equivalent amino acid sequencemay be used in the practice of the method of the invention. Suchalterations of Flt4 nucleotide sequences include deletions, additions orsubstitutions of different nucleotides resulting in a sequence thatencodes the same or a functionally equivalent gene product. The geneproduct may contain deletions, additions or substitutions of amino acidresidues within the sequence which result in silent changes thusproducing a bioactive product. Such amino acid substitutions may be madeon the basis of similarity in polarity, charge, solubility,hydrophobicity, hydrophilicity and/or the amphipathic nature of theresidues involved. For example, negatively charged amino acids includeaspartic acid and glutamic acid; positively charged amino acids includelysine and arginine; amino acids with uncharged polar head groups ornonpolar head groups having similar hydrophilicity values include thefollowing: leucine, isoleucine, valine; glycine, alanine; asparagine,glutamine; serine, threonine; phenylalanine, tyrosine.

Construction of Flt4 Expression Vectors

Using this information, a variety of recombinant DNA vectors capable ofproviding the Flt4 receptor tyrosine kinase in reasonable quantities areprovided. Additional recombinant DNA vectors of related structure thatcode for synthetic proteins having the key structural featuresidentified herein as well as for proteins of the same family from othersources can be produced from the Flt4 receptor tyrosine kinase cDNAusing standard techniques of recombinant DNA technology. A transformantexpressing the Flt4 receptor tyrosine kinase has been produced as anexample of this technology (see EXAMPLES 3 and 4). The newly discoveredsequence and structure information can be used, through transfection ofeukaryotic cells, to prepare the Flt4 receptor tyrosine kinase and itsvarious domains for biological purposes.

Identification of Transfectants or Transformants Expressing Flt4 GeneProducts

The host cells which contain the recombinant coding sequence and whichexpress the biologically active, mature product may be identified by atleast four general approaches: (a) DNA-DNA, DNA-RNA or RNA-antisense RNAhybridization; (b) the presence or absence of “marker” gene functions;(c) assessing the level of transcription as measured by the expressionof Flt4 mRNA transcripts in the host cell; and (d) detection of themature gene product as measured by immunoassay and, ultimately, by itsbiological activities.

In the first approach, the presence of Flt4 coding sequences insertedinto expression vectors may be detected by DNA-DNA hybridization usingprobes comprising nucleotide sequences that are homologous to the Flt4coding sequence.

In the second approach, the recombinant expression vector/host systemmay be identified and selected based upon the presence or absence ofcertain “marker” gene functions (e.g., thymidine kinase activity,resistance to antibiotics, resistance to methotrexate, transformationphenotype, occlusion body formation in baculovirus, etc.). For example,if the Flt4 coding sequence is inserted within a marker gene sequence ofthe vector, recombinants containing that coding sequence can beidentified by the absence of the marker gene function. Alternatively, amarker gene can be placed in tandem with the Flt4 sequence under thecontrol of the same or different promoter used to control the expressionof the Flt4 coding sequence. Expression of the marker in response toinduction or selection indicates expression of the Flt4 coding sequence.

In the third approach, transcriptional activity for the Flt4 codingregion may be assessed by hybridization assays. For example,polyadenylated RNA can be isolated and analyzed by Northern blottingusing a probe homologous to the Flt4 coding sequence or particularportions thereof. Alternatively, total nucleic acids of the host cellmay be extracted and assayed for hybridization to such probes.

In the fourth approach, the expression of Flt4 can be assessedimmunologically, for example by Western blots, immunoassays such asradioimmunoprecipitation, enzyme-linked immunoassays and the like. Theultimate test of the success of the expression system, however, involvesthe detection of the biologically active Flt4 gene product. Where thehost cell secretes the gene product, the cell free media obtained fromthe cultured transfectant host cell may be assayed for Flt4 activity.Where the gene product is not secreted, cell lysates may be assayed forsuch activity. In either case, assays which measure ligand binding toFlt4 or other bioactivities of Flt4 may be used.

Flt4 Derivatives, Analogues and Peptides

The production and use of derivatives, analogues, and peptides relatedto Flt4 are also envisioned and are within the scope of the invention.Such derivatives, analogues, or peptides may have enhanced or diminishedbiological activities in comparison to native Flt4, depending on theparticular application. Flt4 related derivatives, analogues, andpeptides of the invention may be produced by a variety of means known inthe art. Procedures and manipulations at the genetic and protein levelsare within the scope of the invention. Peptide synthesis, which isstandard in the art, may be used to obtain Flt4 peptides. At the proteinlevel, numerous chemical modifications may used to produce Flt4-likederivatives, analogues, or peptides by techniques known in the art,including but not limited to specific chemical cleavage byendopeptidases (e.g. cyanogen bromides, trypsin, chymotrypsin, V8protease, and the like) or exopeptidases, acetylation, formylation,oxidation, etc.

Preferred derivatives, analogs, and peptides are those which retain Flt4ligand binding activity. Those derivatives, analogs, and peptides whichbind Flt4 ligand but do not transduce a signal in response thereto areuseful as Flt4 inhibitors. Those derivatives, analogs, and peptideswhich bind Flt4 ligand and transduce a signal in response thereto, e.g.,through a process involving intracellular Flt4 autophosphorylation, areuseful in the same manner as native Flt4. A preferred Flt4 ligand foruse in such binding and/or autophosphorylation assays is a ligandcomprising an approximately 23 kd polypeptide that is isolatable from aPC-3 conditioned medium as described herein. This ligand, designatedVascular Endothelial Growth Factor-C (VEGF-C), has been characterized indetail in PCT Patent Application PCT/FI96/00427, filed Aug. 1, 1996, andpublished as International Publication WO 97/05250, and in the U.S.patent application priority documents relied upon therein for priority,all of which are incorporated herein by reference in their entirety.

Anti-Flt4 Antibodies

Also within the scope of the invention is the production of polyclonaland monoclonal antibodies which recognize Flt4 or related proteins.

Various procedures known in the art may be used for the production ofpolyclonal antibodies to epitopes of Flt4. For the production ofantibodies, various host animals (including but not limited to rabbits,mice, rats, etc.) can be immunized by injection with Flt4, or asynthetic Flt4 peptide. Various adjuvants may be used to increase theimmunological response, depending on the host species, including but notlimited to Freund's (complete and incomplete) adjuvant, mineral gelssuch as aluminium hydroxide, surface active substances such aslysolecithin, pluronic polyols, polyanions, oil emulsions, keyholelimpet hemocyanins, dinitrophenol, and potentially useful humanadjuvants such as BCG (Bacillus Calmette-Guerin) and Corynebacteriumparvum.

A monoclonal antibody to an epitope of Flt4 may be prepared by using anytechnique which provides for the production of antibody molecules bycontinuous cell lines in culture. These include but are not limited tothe hybridoma technique originally described by Köhler et al., Nature,256: 495-497 (1975), and the more recent human B-cell hybridomatechnique [Kosbor et al., Immunology Today, 4: 72 (1983)] and theEBV-hybridoma technique [Cole et al., Monoclonal Antibodies and CancerTherapy, Alan R Liss, Inc., pp. 77-96 (1985)]. Antibodies against Flt4also may be produced in bacteria from cloned immunoglobulin cDNAs. Withthe use of the recombinant phage antibody system it may be possible toquickly produce and select antibodies in bacterial cultures and togenetically manipulate their structure.

Antibody fragments which contain the idiotype of the molecule may begenerated by known techniques. For example, such fragments include butare not limited to: the F(ab′)₂ fragment which may be produced by pepsindigestion of the antibody molecule; the Fab′-fragments which may begenerated by reducing the disulfide bridges of the F(ab′)₂ fragment, andthe two Fab fragments which may be generated by treating the antibodymolecule with papain and a reducing agent.

Antibodies to Flt4 may be used in the qualitative and quantitativedetection of mature Flt4 and Flt4 precursor and subcomponent forms, inthe affinity purification of Flt4 polypeptides, and in the elucidationof Flt4 biosynthesis, metabolism and function. Detection of Flt4tyrosine kinase activity may be used as an enzymatic means of generatingand amplifying a Flt4 specific signal in such assays. Antibodies to Flt4may also be useful as diagnostic and therapeutic agents.

Uses of Flt4, Flt4-Encoding Nucleic Acid Molecules and Anti-Flt4Antibodies

Applicants envision a wide variety of uses for the compositions of thepresent invention, including diagnostic and/or therapeutic uses of Flt4,Flt4 analogues and derivatives, Flt4-encoding nucleic acid molecules,antisense nucleic acid molecules and anti-Flt4 antibodies.

Flt4-encoding nucleic acid molecules or fragments thereof may be used asprobes to detect and quantify mRNAs encoding Flt4. Assays which utilizenucleic acid probes to detect sequences comprising all or part of aknown gene sequence are well known in the art. Flt4 mRNA levels mayindicate emerging and/or existing neoplasias as well as the onset and/orprogression of other human diseases. Therefore, assays which can detectand quantify Flt4 mRNA may provide a valuable diagnostic tool.

Anti-sense Flt4 RNA molecules are useful therapeutically to inhibit thetranslation of Flt4-encoding mRNAs where the therapeutic objectiveinvolves a desire to eliminate the presence of Flt4 or to downregulateits levels. Flt4 anti-sense RNA, for example, could be useful as a Flt4antagonizing agent in the treatment of diseases in which Flt4 isinvolved as a causative agent, for example due to its overexpression.

Additionally, Flt4 anti-sense RNAs are useful in elucidating Flt4functional mechanisms. Flt4-encoding nucleic acid molecules may be usedfor the production of recombinant Flt4 proteins and related molecules asseparately discussed in this application.

Anti-Flt4 antibodies may be used to diagnose and quantify Flt4 invarious contexts. For example, antibodies against various domains ofFlt4 may be used as a basis for Flt4 immunoassays or immunohistochemicalassessment of Flt4. Tyrosine kinase activity of Flt4 may be useful inthese assays as an enzymatic amplification reaction for the generationof a Flt4 signal. Anti-Flt4 antibodies may also be useful in studyingthe amount of Flt4 on cell surfaces.

Antibodies may be produced which function as Flt4 ligand agonists orantagonists whereby the regulation of Flt4 activity becomes possible.Also, random peptides may be produced by synthetic means or byrecombinant means from random oligonucleotides and the ones showingspecific binding to the Flt4 receptor may be selected with the aid ofthe Flt4 extracellular domain. Such peptide segments also may beselected from a phage display library using the extracellular domain ofFlt4, using methods standard in the art. Such peptides may haveagonistic or antagonistic activity. Flt4 antibodies may also providevaluable diagnostic tools after conjugation to various compounds for invivo imaging of Flt4 expressing cells and tissues or tumors.

Monoclonal antibodies against Flt4 may be coupled either covalently ornoncovalently to a suitable supramagnetic, paramagnetic, electron-dense,echogenic or radioactive agent to produce a targeted imaging agent.Antibody fragments generated by proteolysis or chemical treatments ormolecules produced by using the epitope binding domains of themonoclonal antibodies could be substituted for the intact antibody. Thisimaging agent would then serve as a contrast reagent for X-ray, magneticresonance, sonographic or scintigraphic imaging of the human body fordiagnostic purposes.

Molecular Biology of Flt4

The complete sequences of the Flt4 cDNA clones set forth in SEQ ID NOs:1 and 3 extend for 4195 or 4795 nucleotides and contain open readingframes of 1298 or 1363 amino acids, depending on alternative splicing.The nucleotide and deduced Flt4 amino acid sequence (short form) isshown in SEQ ID NOs: 1 and 2. FIG. 2 depicts a comparison of the Flt4amino acid sequence with that of the Flt1 tyrosine kinase amino acidsequence. See Shibuya et al., Ozcogene, 5: 519-524 (1990).

A putative signal peptide sequence of mostly hydrophobic amino acidsfollows the initiator methionine. The sequence surrounding thecorresponding ATG is in agreement with the consensus translationinitiation sequence [Kozak, Nucl. Acids Res., 15: 8125-8135 (1987)]. Thepredicted extracellular portion of both Flt4 polypeptides is 775 aminoacids long and contains twelve potential sites for asparagine-linkedglycosylation (NXS/T). It also contains several amino acid residuesexhibiting a pattern of spacing described for members of theimmunoglobulin superfamily of proteins [Williams et al., Annu. Rev.Immunol., 6: 381-405 (1988)]. It has 12 cysteine residues and it can beorganized in seven immunoglobulin-like domains. As shown in FIG. 2, theseven immunoglobulin-like domains are defined approximately as follows:Ig-I (SEQ ID NO: 1, positions 158-364; SEQ ID NO: 2, amino acids47-115); Ig-II (SEQ ID NO: 1, positions 479-649; SEQ ID NO: 2, aminoacids 154-210); Ig-III (SEQ ID NO: 1, positions 761-961; SEQ ID NO: 2,amino acids 248-314); Ig-IV (SEQ ID NO: 1, positions 1070-1228; SEQ IDNO: 2, amino acids 351-403); Ig-V (SEQ ID NO: 1, positions 1340-1633;SEQ ID NO: 2, amino acids 441-538); Ig-VI (SEQ ID NO: 1, positions1739-1990; SEQ ID NO: 2, amino acids 574-657); and Ig-VII (SEQ ID NO: 1,positions 2102-2275; SEQ ID NO: 2, amino acids 695-752). The predictedIg-like domain IV lacks cysteine residues. FIG. 2 also shows theextracellular domain of Flt1 (SEQ. ID No. 5), which is the closest humanhomologue of Flt4. From this figure one can see the alignment of thecysteine residues and the very similar composition of the Ig-likeregions.

The cytoplasmic domain of Flt4 is separated from the extracellular partby a putative transmembrane region of 23 hydrophobic amino acidresidues. This sequence is flanked on the cytoplasmic side by a basicregion suggesting the junction between the transmembrane and cytoplasmicdomains. The tyrosine kinase homologous domain begins at residue 843 andincludes an ATP-binding pocket and a putative autophosphorylation sitehomologous to Y416 of c-src at Y1068 (FIG. 2). The tyrosine kinasecatalytic domain of Flt4 is divided into two subdomains by a 65 aminoacid sequence (aa 944-1008) which is mostly hydrophilic and does notshow homology to Flt1. Unlike Flt1, Flt4 does not contain tyrosineresidues in its kinase insert.

A second species of Flt4 mRNA has an alternative 3′ end which encodes alonger form of the Flt4 protein.

In FIGS. 3A-C, production of short and long forms of the Flt4 mRNA byalternative splicing is illustrated. FIG. 3A shows the schematicstructure of the 3′ ends of the cDNA inserts of clones J.1.1 and I.1.1.The TAG stop codon of clone J.1.1 as well as the polyadenylation site(polyA) are indicated. Clone I.1.1 differs from clone J.1.1 in theshaded segment (the long and short forms of Flt4 mRNA, respectively).TAA and polyA indicate the stop codon and polyadenylation site of cloneI.1.1.1. In addition, the restriction endonuclease cleavage sites forEcoRI and AvaI have been indicated. Shown below is the 256 bp EcoRI-AvaIinsert of clone I.1.1. used for cRNA protection analysis. The mostheavily-shaded segment indicates sequences from the polylinker in thelinearized sense RNA template for transcription of the antisense strandin vitro. Also shown are the schematic structures of the protectedfragments after RNAse protection analysis. FIGS. 3B and 3C, showautoradiograms of the 256 bp ³⁵S-labeled antisense RNA probe and the 211and 124 bp digested fragments representing the long and short forms ofFlt4 RNA when protected by polyadenylated RNA from the indicated celllines (Tera-2 is a teratocarcinoma cell line, which has been analyzedhere with or without retinoic acid (RA) treatment for 10 days.) The(negative) control lane shows results of protection with transfer RNA.Note the downregulation of Flt4 mRNAs during the differentiation of theTera-2 cells. Tera-2 cells of clone 13 were provided by Dr. C. F. Graham(Department of Zoology, University of Oxford, UK). Cells betweenpassages 18-40 were used in this study. The cells were maintained inEagle's minimum essential medium (MEM) supplemented with 10% fetal calfserum and antibiotics. To induce differentiation, the cells were platedon gelatin-coated tissue-culture grade dishes at a density of 1.5×10³cells/cm². On the following day, 2×10⁻⁶ M RA was added to the medium.The cells were cultured in the presence of RA for up to 10 days.

Results shown in FIGS. 3A-C illustrate the generation of carboxy terminiof these two Flt4 (short and long) forms generated by alternativesplicing.

According to its deduced amino acid sequence, Flt4 belongs to class IIIRTKs. More specifically, Flt4 belongs to a subfamily of RTKs, whichcontain seven Ig-loops in their extracellular part and thus it differsfrom other members of class III RTKs which contain five Ig-loops. Flt4is most closely homologous with the prototype receptor of the FLTfamily, Flt1, which was cloned as a v-ros-related DNA from a humangenomic DNA library [Shibuya et al., Oncogene, 5: 519-524 (1990)] andwith the mouse FLK1 receptor, which was cloned from hematopoietic stemcell-enriched fractions of mouse liver [Matthews et al., Cell, 65:1143-1152 (1991); Matthews et al., Proc. Natl. Acad. Sci. USA, 88:9026-9030 (1991)]. The extracellular domain of Flt4 shows 33% and 37%amino acid sequence identity with human Flt1 and mouse FLK1,respectively. Flt1 and FLK1, like Flt4, are widely expressed in variousnormal tissues, such as lung, heart, and kidney. In addition, a recentlyidentified human endothelial cell receptor tyrosine kinase KDR [Termanet al., Oncogene, 6: 1677-1683 (1991)] shows considerable homology withFlt4 and Flt1 family members. From the available sequence data one maycalculate that KDR is 81% identical with Flt4 in the tyrosine kinase(TK) domain. In addition, the extracellular domain of KDR also has aseven Ig-loop structure and its TK1 and TK2 domains are 95% and 97%identical with the corresponding domains of mouse FLK1 receptor. Thissuggests that KDR is the human homologue of mouse FLK1.

While the Flt4 TK domain is about 80% identical with the TK domains ofFlt1 and FLK1/KDR, it is only about 60% identical with the TK domains ofother receptors of the RTK class III. As these other receptors also haveonly five Ig-like domains in the extracellular region, one can classifyFlt4, Flt1 and FLK1/KDR in a separate FLT subfamily within class IIIRTKs.

The tyrosine residue located in the sequence D/E-D/E-Y-M/V-P/D/E-M[Cantley, et al., Cell, 64: 281-302 (1991)] (SEQ. ID NO. 6) in kinaseinserts of PDGFRs, c-fms and c-kit is an autophosphorylation site,which, when phosphorylated, binds the SH2 domain of phosphatidylinositol3′-kinase (PI-3K) [Reedijk et al., EMBO J, 11: 1365-1372 (1992)].Interestingly, unlike these class III RTKs, members of the FLT subfamilyor the Flt3/FLK2 receptor do not contain such consensus motifs.

The eight human class III RTK genes are clustered in three differentchromosomes. Chromosome 4 contains the c-kit, PDGFR-α and KDR genes[Yarden et al., EMBO J, 6: 3341-3351 (1987); Stenman et al., Genes,Chromosomes, Cancer, 1: 155-158 (1989); Terman et al., Oncogene,6:1677-1683 (1991)]. The Flt1 and Flt3 genes are located in chromosome13q12 [Satoh et al., Jpn. J. Cancer Res., 78: 772-775 (1987); Rosnet etal., Genomics, 9: 380-385 (1991)], while Flt4 is localized in chromosome5 band q35 [Aprelikova et al., Cancer Res., 52: 746-748 (1992)]; closeto the fms and PDGFR-β genes [Warrington et al., Genomics, 11: 701-708(1991). The long arm of chromosome 5 is involved in translocations foundin leukemia cells. Deletions of part of the long arm of chromosome 5were found in the bone marrow cells of patients with refractory anemiaand macrocytosis [Van Den Berghe et al., Nature, 251: 437-439 (1974)].An abnormal 5q chromosome is found in a few other myeloproliferativediseases, such as refractory anemia with excess blasts [Swolin et al.,Blood, 58: 986-993 (1981)], agnogenic myeloid metaplasia [Whang-Peng etal., Leuk. Res., 2: 41-48 (1978)], chronic myelogenous leukemia[Tomiyasu et al., Cancer Genet. Cytogenet., 2: 309-315 (1980)],polycythemia vera [Van Den Berghe et al., Cancer Genet. Cytogenet., 1:157-162 (1979)] and essential thrombocythemia [Nowell et al., Cancer,42: 2254-2260 (1978)].

The findings on Flt4 mRNA expression suggest that its protein product ischaracteristic for certain leukemia cells. Several differentiationantigens shared between megakaryoblastic and endothelial cells have beenshown to exist, one example being the platelet glycoprotein IIIa [Ylänneet al., Blood, 72: 1478-1486 (1988); Kieffer et al., Blood, 72:1209-1215 (1988); Berridge et al., Blood, 66: 76-85 (1985)]. Inaddition, Flt4 is expressed by certain endothelial cells of, e.g., thelung and kidney during the fetal period.

To further understand the role of Flt4 during development, partial cDNAsfor mouse Flt4 were cloned. Using these probes in in situ hybridization,Flt4 mRNA expression during mouse development was analyzed. It wasdetermined that Flt4 is expressed during vasculogenesis and angiogenesisof the lymphatic system. The relevance of these fingings was alsoconfirmed in normal and pathological human adult tissues, as Flt4 wasfound in lymphatic endothelial cells of human adult tissues both innormal and pathological conditions, as well as in some high endothelialvenules (HEVs).

The cloning of mouse Flt4 cDNA fragments showed that their deduced aminoacid sequence is almost identical with the corresponding human sequence(amino acid identity about 96% in both segments studied). Furtherevidence for the identity of the mouse Flt4 cDNA was obtained fromNorthern hybridization studies, wherein probes from both species yieldedthe typical 5.8 kb mRNA signal from mouse tissues. Analysis of RNAisolated from various tissues of adult mice showed Flt4 expression inthe liver, lung, heart, spleen and kidney, with no or very littlehybridization in the brain and testes. This pattern is similar to thepattern reported earlier by Galland et al., Oncogene, 8: 1233 (1993).The results of RNase protection suggested that the Flt4 gene is neededduring mouse development, starting from 8.5 day p.c. embryos, and therelative expression levels appeared quite stable.

For the in situ hybridization, two fragments of mouse Flt4 cDNA wereselected which encode sequences of the extracellular domain. Thisallowed a clear distinction of the hybridization pattern from therelated FLK-1 and Flt1 receptor patterns, which show only a very lowdegree of sequence identity with Flt4 in the extracellular region. SeeMillauer et al., Cell, 72: 835 (1993); Yamaguchi et al., Development,118:489 (1993); Peters et al., Proc. Natl. Acad. Sci. USA, 90: 8915(1993); Finnerty et al., Oncogene, 8: 2293 (1993).

Flt4, similar to the FLK-1, Flt1, Tie and Tek endothelial receptortyrosine kinase genes, was not expressed in 7.5 day post-coitum (p.c.)embryos. In a 8.5-day p.c. embryo, the strongest Flt4 signals werelocalised in the allantois, the angioblasts of head mesenchyme, thedorsal aortae, and the cardinal vein. Weak signals were seen in theendocardium. In contrast, angioblasts of the yolk sac were negative,unlike for FLK-1 and Flt1, Tie and Tek. See Korhonen et al., Oncogelle.8: 395 (1993); and Peters et al., Proc. Natl. Acad. Sci. USA, 90: 8915(1993). The restriction of Flt4 expression to the venous system was evenmore clear in samples from 11.5 day mouse embryos, where the Tie mRNAwas expressed also in arteries. In 12.5-day p.c. embryos the Flt4 signaldecorated developing venous and presumptive lymphatic endothelia, butunlike for the endothelial Tie receptor tyrosine kinase, arterialendothelia were negative. During-later stages of development, Flt4 mRNAbecame restricted to vascular plexuses devoid of blood cells,representing developing lymphatic vessels. Only the lymphaticendothelium and some high endothelial venules expressed Flt4 mRNA inadult human tissues. Increased expression occurred in lymphatic sinusesand high endothelial venules, in metastatic lymph nodes, and inlymphangioma.

Due to difficulties in the interpretation of data from mouse embryos,human endothelia were studied, because the lymphatic system is muchbetter defined in humans. Also, cells established from variousendothelia could be studied in cell culture to see if the specificity ofFlt4 expression persists in in vitro conditions. Endothelial cells linesare known to lose differentiated features upon in vitro culture.Therefore, it was not unexpected that they were negative for Flt4 mRNA.Cultured aortic endothelial cells were also devoid of Flt4 mRNA.However, signals were obtained from human endothelial cells grown fromthe microvasculature and from femoral and umbilical veins. Thus, atleast some of the specificity of Flt4 expression was retained in cellculture.

In situ hybridization analysis of adult human tissues confirmed therestriction of Flt4 to the lymphatic system seen in the developing mouseembryos. Flt4 expression was seen in the lymphatic endothelia and in thesinuses of human lymph nodes. Interestingly, also some of the HEVs,which have a cuboidal endothelium, shown to function in the traffickingof leukocytes to the lymph nodes, were Flt4-positive. Furthermore, aparallel hybridization analysis showed that Flt4 mRNA levels wereenhanced in these structures in metastatic as compared to normal lymphnodes. Flt4 was also very prominent in lymphangiomas, which are benigntumours composed of connective tissue stroma and growing,endothelial-lined lymphatic channels. Flt4 mRNA was restricted to thelymphatic endothelium of these tumors and absent, from their arteries,veins and capillaries. In the human lung, lymphatic structures were theonly Flt4-positive vessels identified.

The foregoing results indicate that Flt4 is a novel marker for lymphaticvessels and some high endothelial venules in human adult tissues. Theresults also support the theory on the venous origin of lymphaticvessels. Flt4, as a growth factor receptor, may be involved in thedifferentiation and functions of these vessels. A detailedcharacterization of biological effects mediated through Flt4 via theFlt4 ligand, VEGF-C, is provided in PCT Patent ApplicationPCT/FI96/00427, filed Aug. 1, 1996, and published as InternationalPublication WO 97/05250.

These results, combined with the Flt4-binding compounds according to thepresent invention, allows a selective labeling of lymphatic endothelium,especially by using antibodies of the present invention coupled toradioactive, electron-dense or other reporter substances, which can bevisualized. It may be possible to inject into the lymphatic systemsubstances, containing Flt4 receptor internalization-inducing monoclonalantibodies or ligands, and thereby transport predefined molecules intothe lymphatic endothelium. Also, it may be possible to use Flt4-bindingcompounds according to the invention for the detection of highendothelial venules, especially activated HEVs, which express enhancedlevels of the Flt4 receptor. To our knowledge, no such specific markersare currently available for lymphatic endothelium.

Gene Based Therapies

The present invention also contemplates gene therapy methods.Specifically, the vasculature of the cancer cell or the cancer cellitself may be contacted with an expression construct capable ofproviding a therapeutic peptide, such as for example, the solubleVEGFR-3 fragments of the present invention, to the vasculature of thecell in a manner to effect a therapeutic outcome. Such a therapeuticoutcome may be, for example, inhibition of VEGFR-3 in the vasculature ofthe tumor, an inhibition of angiogensis, an inhibition oflymphangiogenesis, an abalattion, regression or other inhibition oftumor growth, an induction of apoptosis of the blood or lymphaticvasculature of the tumor or indeed the tumor cells themselves.

For these embodiments, an exemplary expression construct comprises avirus or engineered construct derived from a viral genome. Theexpression construct generally comprises a nucleic acid encoding thegene to be expressed and also additional regulatory regions that willeffect the expression of the gene in the cell to which it isadministered. Such regulatory regions include for example promoters,enhancers, polyadenylation signals and the like.

It is now widely recognized that DNA may be introduced into a cell usinga variety of viral vectors. In such embodiments, expression constructscomprising viral vectors containing the genes of interest may beadenoviral (see for example, U.S. Pat. No. 5,824,544; U.S. Pat. No.5,707,618; U.S. Pat. No. 5,693,509; U.S. Pat. No. 5,670,488; U.S. Pat.No. 5,585,362; each incorporated herein by reference), retroviral (seefor example, U.S. Pat. No. 5,888,502; U.S. Pat. No. 5,830,725; U.S. Pat.No. 5,770,414; U.S. Pat. No. 5,686,278; U.S. Pat. No. 4,861,719 eachincorporated herein by reference), adeno-associated viral (see forexample, U.S. Pat. No. 5,474,935; U.S. Pat. No. 5,139,941; U.S. Pat. No.5,622,856; U.S. Pat. No. 5,658,776; U.S. Pat. No. 5,773,289; U.S. Pat.No. 5,789,390; U.S. Pat. No. 5,834,441; U.S. Pat. No. 5,863,541; U.S.Pat. No. 5,851,521; U.S. Pat. No. 5,252,479 each incorporated herein byreference), an adenoviral-adenoassociated viral hybrid (see for example,U.S. Pat. No. 5,856,152 incorporated herein by reference) or a vacciniaviral or a herpesviral (see for example, U.S. Pat. No. 5,879,934; U.S.Pat. No. 5,849,571; U.S. Pat. No. 5,830,727; U.S. Pat. No. 5,661,033;U.S. Pat. No. 5,328,688 each incorporated herein by reference) vector.

In other embodiments, non-viral delivery is contemplated. These includecalcium phosphate precipitation (Graham and Van Der Eb, Virology,52:456-467, 1973; Chen and Okayama, Mol. Cell Biol., 7:2745-2752, 1987;Rippe et al., Mol. Cell Biol., 10:689-695, 1990) DEAE-dextran (Gopal,Mol. Cell Biol., 5:1188-1190, 1985), electroporation (Tur-Kaspa et al.,Mol. Cell Biol., 6:716-718, 1986; Potter et al., Proc. Nat. Acad. Sci.USA, 81:7161-7165, 1984), direct microinjection (Harland and Weintraub,J. Cell Biol., 101:1094-1099, 1985.), DNA-loaded liposomes (Nicolau andSene, Biochim. Biophys. Acta, 721:185-190, 1982; Fraley et al., Proc.Natl. Acad. Sci. USA, 76:3348-3352, 1979; Feigner, Sci Am. 276(6):102-6,1997; Feigner, Hum Gene Ther. 7(15):1791-3, 1996), cell sonication(Fechheimer et al., Proc. Natl. Acad. Sci. USA, 84:8463-8467, 1987),gene bombardment using high velocity microprojectiles (Yang et al.,Proc. Natl. Acad. Sci USA, 87:9568-9572, 1990), and receptor-mediatedtransfection (Wu and Wu, J. Biol. Chem., 262:4429-4432, 1987; Wu and Wu,Biochemistry, 27:887-892, 1988; Wu and Wu, Adv. Drug Delivery Rev.,12:159-167, 1993).

In a particular embodiment of the invention, the expression construct(or indeed the peptides discussed above) may be entrapped in a liposome.Liposomes are vesicular structures characterized by a phospholipidbilayer membrane and an inner aqueous medium. Multilamellar liposomeshave multiple lipid layers separated by aqueous medium. They formspontaneously when phospholipids are suspended in an excess of aqueoussolution. The lipid components undergo self-rearrangement before theformation of closed structures and entrap water and dissolved solutesbetween the lipid bilayers (Ghosh and Bachhawat, In: Liver diseases,targeted diagnosis and therapy using specific receptors and ligands, WuG, Wu C ed., New York: Marcel Dekker, pp. 87-104, 1991). The addition ofDNA to cationic liposomes causes a topological transition from liposomesto optically birefringent liquid-crystalline condensed globules (Radleret al., Science, 275(5301):810-4, 1997). These DNA-lipid complexes arepotential non-viral vectors for use in gene therapy and delivery.

Liposome-mediated nucleic acid delivery and expression of foreign DNA invitro has been very successful. Also contemplated in the presentinvention are various commercial approaches involving “lipofection”technology. In certain embodiments of the invention, the liposome may becomplexed with a hemagglutinating virus (HVJ). This has been shown tofacilitate fusion with the cell membrane and promote cell entry ofliposome-encapsulated DNA (Kaneda et al., Science, 243:375-378, 1989).In other embodiments, the liposome may be complexed or employed inconjunction with nuclear nonhistone chromosomal proteins (HMG-1) (Katoet al., J. Biol. Chem., 266:3361-3364, 1991). In yet furtherembodiments, the liposome may be complexed or employed in conjunctionwith both HVJ and HMG-1. In that such expression constructs have beensuccessfully employed in transfer and expression of nucleic acid invitro and in vivo, then they are applicable for the present invention.

Other vector delivery systems that can be employed to deliver a nucleicacid encoding a therapeutic gene into cells include receptor-mediateddelivery vehicles. These take advantage of the selective uptake ofmacromolecules by receptor-mediated endocytosis in almost all eukaryoticcells. Because of the cell type-specific distribution of variousreceptors, the delivery can be highly specific (Wu and Wu, 1993, supra).

Receptor-mediated gene targeting vehicles generally consist of twocomponents: a cell receptor-specific ligand and a DNA-binding agent.Several ligands have been used for receptor-mediated gene transfer. Themost extensively characterized ligands are asialoorosomucoid (ASOR) (Wuand Wu, 1987, supra) and transferrin (Wagner et al., Proc. Nat'l. Acad.Sci. USA, 87(9):3410-3414, 1990). Recently, a synthetic neoglycoprotein,which recognizes the same receptor as ASOR, has been used as a genedelivery vehicle (Ferkol et al., FASEB J., 7:1081-1091, 1993; Perales etal., Proc. Natl. Acad. Sci., USA 91:40864090, 1994) and epidermal growthfactor (EGF) has also been used to deliver genes to squamous carcinomacells (Myers, EPO 0273085).

In other embodiments, the delivery vehicle may comprise a ligand and aliposome. For example, Nicolau et al. (Methods Enzymol., 149:157-176,1987) employed lactosyl-ceramide, a galactose-terminal asialganglioside,incorporated into liposomes and observed an increase in the uptake ofthe insulin gene by hepatocytes. Thus, it is feasible that a nucleicacid encoding a therapeutic gene also may be specifically delivered intoa particular cell type by any number of receptor-ligand systems with orwithout liposomes.

In another embodiment of the invention, the expression construct maysimply consist of naked recombinant DNA or plasmids. Transfer of theconstruct may be performed by any of the methods mentioned above thatphysically or chemically permeabilize the cell membrane. This isapplicable particularly for transfer in vitro, however, it may beapplied for in vivo use as well. Dubensky et al. (Proc. Nat. Acad. Sci.USA, 81:7529-7533, 1984) successfully injected polyomavirus DNA in theform of CaPO₄ precipitates into liver and spleen of adult and newbornmice demonstrating active viral replication and acute infection.Benvenisty and Neshif (Proc. Nat. Acad. Sci. USA, 83:9551-9555, 1986)also demonstrated that direct intraperitoneal injection of CaPO₄precipitated plasmids results in expression of the transfected genes.

Another embodiment of the invention for transferring a naked DNAexpression construct into cells may involve particle bombardment. Thismethod depends on the ability to accelerate DNA coated microprojectilesto a high velocity allowing them to pierce cell membranes and entercells without killing them (Klein et al., Nature, 327:70-73, 1987).Several devices for accelerating small particles have been developed.One such device relies on a high voltage discharge to generate anelectrical current, which in turn provides the motive force (Yang etal., Proc. Natl. Acad. Sci USA, 87:9568-9572, 1990). Themicroprojectiles used have consisted of biologically inert substancessuch as tungsten or gold beads.

Those of skill in the art are well aware of how to apply gene deliveryto in vivo and ex vivo situations. For viral vectors, one generally willprepare a viral vector stock. Depending on the kind of virus and thetiter attainable, one will deliver 1×10⁴, 1×10⁵, 1×10 ⁶, 1×10⁷, 1×10⁸,1×10⁹, 1×10¹⁰, 1×10¹¹ or 1×10¹² infectious particles to the patient.Similar figures may be extrapolated for liposomal or other non-viralformulations by comparing relative uptake efficiencies. Formulation as apharmaceutically acceptable composition is discussed below.

Various routes are contemplated for various tumor types. The sectionbelow on routes contains an extensive list of possible routes. Forpractically any tumor, systemic delivery is contemplated. This willprove especially important for attacking microscopic or metastaticcancer. Where discrete tumor mass may be identified, a variety ofdirect, local and regional approaches may be taken. For example, thetumor may be directly injected with the expression vector or protein. Atumor bed may be treated prior to, during or after resection. Followingresection, one generally will deliver the vector by a catheter left inplace following surgery. One may utilize the tumor vasculature tointroduce the vector into the tumor by injecting a supporting vein orartery. A more distal blood supply route also may be utilized.

In a different embodiment, ex vivo gene therapy is contemplated. In anex vivo embodiment, cells from the patient are removed and maintainedoutside the body for at least some period of time. During this period, atherapy is delivered, after which the cells are reintroduced into thepatient; preferably, any tumor cells in the sample have been killed.

The following examples are given merely to illustrate the presentinvention and not in any way to limit its scope.

EXAMPLE 1 Isolation and Characterization of cDNA Clones Encoding Flt4Materials and Methods

An oligo-dT primed human HEL cell cDNA library in bacteriophage lambdagt11 [A kind gift from Dr. Mortimer Poncz, Childrens Hospital ofPhiladelphia, Pa.; Poncz et al., Blood, 69: 219-223 (1987)] was screenedwith a cDNA fragment PCR-amplified from the same library [Aprelikova etal., Cancer Res., 52: 746-748 (1992)]. Positive plaques were identifiedand purified as described [Sambrook et al., Molecular Cloning—ALaboratory Manual, Cold Spring Harbor Laboratory Press, (1989)]. cDNAinserts of bacteriophage lambda were isolated as EcoRI fragments andsubcloned into a GEM3Zf(+) plasmid (Promega). The entire Flt4 proteincoding region was isolated. Three overlapping clones isolated from theHEL-library (as illustrated in FIG. 1) were sequenced using the dideoxychain termination method with oligonucleotide primers designed accordingto the sequences obtained. All portions of the cDNAs were sequenced onboth strands. Sequence analyses were performed using the GCG packageprograms [Devereux et al., Nucleic Acids Res., 12: 387-395 (1984) andthe Prosite program for Apple MacIntosh].

FIG. 1A illustrates a schematic structure of the Flt4 cDNA clonesanalyzed. Arrows delineate subcloned restriction fragments (whose sizesare shown in kb) used for probing Northern blots depicted in FIG. 1B.E=EcoRI site, S=SphI site. FIG. 1B illustrates Northern hybridizationanalysis of DAMI and HEL leukemia cell RNAs with the probes shown inFIG. 1A.

Results

A 210 bp long Flt4 cDNA fragment isolated by a PCR cloning method from aHEL cell cDNA library was used as a molecular probe to screen anoligo-dT-primed human erythroleukemia cell cDNA library.

Nucleotide sequence analysis of clones revealed an open reading frame of1298 amino acid (aa) residues (SEQ ID NO: 2, FIG. 2). The translationalinitiator methionine marked in the figure is surrounded by a typicalconsensus sequence [Kozak, Nucleic Acids Res., 12: 857-872 (1984)] andfollowed by a hydrophobic amino acid sequence characteristic of signalsequences for translocation into the endoplasmic reticulum.

The extracellular domain of Flt4 can be aligned into sevenimmunoglobulin-like loops (FIG. 2). The figure also shows the comparisonof Flt4 with Fit 1, which contains very similar structures. The aminoacid sequence of Flt1 is set forth as SEQ. ID NO: 5.

Amino acid residues 775-798 form a hydrophobic stretch of sequence,which is likely to function as the transmembrane domain of the receptor,followed by several basic residues on the putative cytoplasmic side ofthe polypeptide. The juxtamembrane domain is 44 residues long before thebeginning of a tyrosine kinase sequence homology at aa 842. With theinterruption of homology in the kinase insert sequence of 65 aa, thishomology is first lost at 1175 aa at carboxyl terminal tail of thereceptor. A search for related tyrosine kinase domains in the amino acidsequence database (Swissprot and NBRF) identifies the Flt1 and PDGFRBtyrosine kinases with homology of about 80 and 60% in the catalytictyrosine kinase regions respectively.

EXAMPLE 2 Preparation of an Anti-Flt4 Antisera

A 657 base pair EcoRI fragment encoding the predicted C-terminus of Flt4short form was cloned in-frame with the glutathione-S-transferase codingregion in the pGEX-1λT bacterial expression vector (Pharmacia) toproduce a GST-Flt4 fusion protein in E. coli. The resulting fusionprotein was produced in bacteria and partially purified by glutathioneaffinity chromatography according to the manufacturer's instructions.This protein was used in immunization of rabbits in order to producepolyclonal antibodies against Flt4. Antisera were used after the thirdbooster immunization.

EXAMPLE 3 Expression of Flt4 in COS Cells Materials and Methods

The full-length Flt4 protein coding sequence (combined from threeclones, FIG. 1) was inserted into the HindIII-BamHI site of SVpolymammalian expression vector [Stacey et al., Nucleic Acids Res., 18: 2829(1990)] construct SV14-2. The expression vectors (SV-FLT4 short andSV-FLT4 long, containing the respective forms of Flt4 cDNA) wereintroduced into COS cells by DEAE-dextran transfection method [McCutchanet al., J. Natl. Cancer Inst., 41: 351-357 (1968)]. Two days aftertransfection, the cells were washed with phosphate-buffered saline (PBS)and scraped into immunoprecipitation buffer (10 mM Tris pH 7.5, 50 mMNaCl, 0.5% sodium deoxycholate, 0.5% Nonidet P40, 0.1% SDS, 0.1 TIU/mlAprotinin). The lysates were sonicated, centrifuged for 15′ at 10,000×gand incubated overnight on ice with 3 ml of the antisera. Protein Asepharose (Pharmacia) was added and the incubation was continued for 30′with rotation. The precipitates were washed four times with theimmunoprecipitation buffer, once with PBS and once with aqua beforeanalysis in SDS-PAGE.

Results

The structural predictions of the Flt4 cDNA sequence were tested bycloning the full-length Flt4 short and long protein-coding regions intothe HindIII-BamHI sites of the pSVpoly expression vector andtransfecting these expression vectors into COS cells. The proteinsproduced by these two constructs differ in their C-terminus: the longerform contains an additional 65 amino acids. Two days after transfection,the cells were lysed and immunoprecipitated using antibodies generatedagainst the GST-Flt4 fusion protein containing 40 carboxyl terminalamino acid residues of the short form of the predicted Flt4 protein(i.e., a portion common to both the short and long forms of Flt4).Immunoprecipitated polypeptides were analyzed by SDS-polyacrylamide gelelectrophoresis. The preimmune serum did not reveal any specific bands,whereas the Flt4-specific antibodies recognize two bands of about 170and 190 KD. These two bands may represent differentially glycosylatedforms of Flt4 protein.

EXAMPLE 4 Expression of Flt4 in NIH3T3 Cells Materials and Methods

The full-length Flt4 cDNA (short form) was subcloned into the LTRpolyvector (see Makela, et al., Gene, 118:293-294 (1992), disclosing plasmidvector pLTRpoly, having ATCC accession number 77109 and GeneBankaccession number X60280) containing the Moloney murine leukemia viruslong terminal repeat promoter. This LTR-FLT4 expression vector was usedwith pSV2neo marker plasmid to co-transfect NIH3T3 cells, and G418resistant clones were analyzed for Flt4 expression.

For Western immunoblotting analyses, cells on one confluent large platewere lysed in 2.5% SDS, 125 mM Tris, pH 6.5. Cell lysates wereelectrophoresed on SDS-page and electroblotted onto a nitrocellulosemembrane. The membrane was incubated with the antiserum raised againstthe Flt4 carboxy-terminus peptide, and bound antibodies were visualizedusing horseradish peroxidase conjugated swine anti-rabbit antiserum(Dakb) and ECL reagents (Amersham). For metabolic labeling, the cultureswere labeled with 100 μCi/ml ³⁵S-methionine for one hour. Afterlabelling, cells were washed twice and incubated in their growth mediumfor 1 or 2 hours, lysed, immunoprecipitated with anti-Flt4 antibodies,and analyzed by SDS-PAGE and autofluorography.

Results

The 170 and 190 KD polypeptides could be detected in the Flt4 shortform-transfected into NIH3T3 cells, but not in cells transfected withpSV2neo only. In addition to these two bands, a major band of about 120Kd was observed in the clones producing Flt4. Metabolic labeling andpulse-chase experiments showed that this protein is generated as aresult of post-translational processing of the short form Flt4polypeptides.

EXAMPLE 5 Chromosomal Mapping of the Flt4 Locus

Because some clustering of class III receptor genes has been observed,it is of great interest to determine the chromosomal localization ofFlt4. Thus, rodent-human cell hybrids were analyzed, indicating linkageof Flt4 to human chromosome 5.

Localization of the Flt4 gene in the region 5q33->5qter was determinedusing hybrids carrying partial chromosome 5s. These hybrids were testedfor presence of the Flt4 locus by filter hybridization. The region ofchromosome 5 common to Flt4-positive hybrids and absent from theFlt4-negative hybrids was 5q33.1-qter. The presence of human chromosome5q33-qter in the hybrids is thus correlated with the presence of Flt4sequences. The regional mapping results indicated that the Flt4 locus istelomeric to the CSF1R/platelet-derived growth factor receptor-β(PDGFRB) locus as well as to the β-adrenergic receptor (ADRBR) locussince these loci are all present in the hybrid GB13, which was negativefor Flt4.

EXAMPLE 6 Expression of the Flt4 mRNA in Tumor Cell Lines andEndothelial Cells

The leukemia cell lines (K562) used in this study have been reported inseveral previous publications; [Lozzio et al., Blood, 45: 321-334(1975)], HL-60 [Collins et al., Nature, 270: 347-349 (1977)], HEL[Martin et al., Science, 216: 1233-1235 (1982)], DAMI [Greenberg et al.,Blood, 72: 1968-1977 (1988)], MOLT-4 [Minowada et al., J. Natl. CancerInst., 49: 891-895 (1972)], Jurkat [Schwenk et al., Blut, 31: 299-306(1975)], U937 [Sundström et al., Int. J. Cancer, 17: 565-577 (1976)],KG-1 [Koeffler et al., Science, 200: 1153-1154 (1978)], JOK-1 [Anderssonet al., 1982, in R. F. Revoltella (ed.), Expression of DifferentiatedFunctions in Cancer Cells, 239-245, Raven Press, New York] and ML-2[Gahmberg et al., 1985, in L. C. Andersson, et al. (ed.), GeneExpression During Normal and Malignant Differentiation, 107-123,Academic Press, London]. The following tumor cell lines, obtained fromthe American Type Culture Collection also were analyzed: JEG-3, achoriocarcinoma; A204, a rhabdomyosarcoma; SK-NEP-1, a nephroblastoma;BT-474, a breast carcinoma; Y79, a retinoblastoma. The leukemia cellswere grown in RPM1 containing 10% fetal calf serum (FCS) andantibiotics. Dami cells were cultivated in Iscove's modified DMEM with10% horse serum. A permanent endothelial hybrid cell line (EAhy926)obtained by fusing first-passage human umbilical vein endothelial cellswith the A549 lung carcinoma cells [Edgell et al., Proc. Natl. Acad.Sci. USA, 50: 3734-3737 (1983)] was cultured in DMEM-HAT mediumcontaining 10% FCS and antibiotics.

Poly(A)⁺ RNA was extracted from the cell lines as described [Sambrook etal., see above]. 5 μg of the Poly(A)⁺ RNA samples were electrophoresedin agarose gels containing formaldehyde and blotted using standardconditions [Sambrook et al., see above]. The inserts of the Flt4 cDNAclones were labelled by the random priming method and hybridized to theblots. Hybridization was carried out in 50% formamide, 5× Denhardt'ssolution (100× Denhardt's solution is 2% each of Ficoll,polyvinylpyrrolidone and bovine serum albumin), 5×SSPE (3M NaCl, 200 mMNaH₂PO₄H₂O, 20 mM EDTA, pH 7.0), 0.1% SDS (sodium dodecyl sulphate), and0.1 mg/ml of sonicated salmon sperm DNA at 42° C. for 18-24 h. Thefilters were washed at 65° C. in 1×SSC (150 mM NaCl, 15 mM sodiumcitrate, pH 7.0), 0.1% SDS and exposed to Kodak XAR-5 film.

Northern analyses were performed with the extracted poly(A)+ RNA fromeight leukemia cell lines (HEL, K562, DAM1, U937, MOLT4, HL60, Jurkat,and KG-1) and the endothelial hybrid cell line (EAhy926). Hybridizationwith the GAPDH probe was used as an internal control for the loading ofeven amounts of RNA to the analysis. Only the HEL erythroleukemia cells,and DAMI megakaryoblastic leukemia cells expressed 5.8 kb and 4.5 kbFlt4 mRNA. The K562 erythroleukemia, Jurkat and MOLT-4 T-cell leukemias,as well as HL-60 promyelocytic leukemia, U937 monocytic leukemia, andKG-1 myeloid leukemia cells were negative for the Flt4 mRNA.

Northern analyses were performed with the extracted poly(A)⁺ RNA fromfive tumor cell lines (JEG-3, A-204, SK-NEP-1, BT-474, and Y79) and twoof the aforementioned leukemia cell lines (JOK-1, MOLT4). The labeledS2.5 cDNA clone (see FIG. 11), was used as the hybridization probe.Hybridization with a β-actin probe was used as an internal control forthe loading of even amounts of RNA to the analysis. Only the SK-NEP-1nefroblastoma and Y79 retinoblastoma cells were observed to contain Flt4transcripts.

Tera-2 teratocarcinoma cells were analyzed after a 10 day treatment withvehicle (−) or retinoic acid (+) to induce neuronal differentiation[Thompson et al., J Cell Sci., 72: 37-64 (1984). In Northern blottinganalysis of poly(A)⁺ RNA isolated from the cells it was found that theundifferentiated cells expressed 5.8 kb and 4.7 kb mRNAs for Flt4, butafter the 10 day differentiation, no Flt4 mRNA could be detected inNorthern blotting and hybridization. These results indicate that Flt4was downregulated during the differentiation of these cells.

Flt4 mRNA expression also was analyzed in undifferentiated andTPA-differentiated HEL cells. Both the HEL and DAMI cell lines possess adual erythroid/megakaryoblastic phenotype and can be induced to furtherexpression of megakaryoblastic markers by treatment with the tumorpromotor 12-O-tetradecanoylphorbol-13-acetate (TPA). We analyzed whetherFlt4 expression is stimulated in these cells during theirdifferentiation. HEL cells were analyzed 2 days after treatment with TPAor with DMSO used to dissolve it. After stripping off the Flt4 signal,the filter was probed with Rb-1 and β-actin cDNAs to confirm an evenloading of the lanes. On the basis of densitometric scanning analysis ofthe autoradiograph and normalization against the constitutive expressionof the GAPDH gene, it was determined that the Flt4 mRNA level wasincreased about 3.4 fold in TPA-induced HEL cells, when the cellsundergo megakaryoblastic differentiation.

EXAMPLE 7 Expression of Flt4 in Fetal Lung

In situ hybridization: Lung tissue from a 15 week-old human fetus wasobtained with the permission of joint ethical committee of theUniversity Central Hospital and the University of Turku, Finland. Thesample was fixed in 10% formalin for 18 hours at 4° C., dehydrated,embedded in wax, and cut into 6 μm sections. The RNA probes of 206 and157 bases (antisense and sense) were generated from linearized plasmidDNA using SP6 and T7 polymerases and [³⁵S]-UTP. In situ hybridization ofsections was performed according to Wilkinson et al., Development,99:493-500 (1987); Wilkinson, Cell, 50:79-88 (1987), with the followingmodifications: 1) instead of toluene, xylene was used before embeddingin paraffin wax; 2) 6 μm sections were cut, placed on a layer of diethylpyrocarbonate-treated water on the surface of glass slides pretreatedwith 2% 3-aminopropyl-triethoxysilane (Sigma); 3) alkaline hydrolysis ofthe probes was omitted; 4) the hybridization mixture contained 60%deionized formamide; 5) the high stringency wash was for 80 minutes at65° C. in a solution containing 50 mM DTT and 1×SSC; 6) the sectionswere covered with NTB-2 emulsion (Kodak) and stored at 4° C. After anexposure time of 14 days, the slides were developed for 2.5 minutes in aKodak D-19 developer and fixed for 5 minutes with Unifix (Kodak). Thesections were stained with hematoxylin in water.

In the hybridization studies using the anti-sense probe, Flt4 mRNA wasobserved mainly in certain endothelial cells of the lungs of a 15 weekold fetus. Control hybridizations with the sense strand probe and withRNAse A-treated sections did not give a signal above background.

For immunoperoxidase staining, a 1:100 dilution of the anti-Flt4antibody, peroxidase-conjugated swine anti-rabbit antibodies and methodsstandard in the art were used. Control stainings with preimmune serum orimmunogen-blocked serum did not give a signal. Lung tissue fromseventeen-week old human fetuses were analyzed, and the results wereconsistent with those of the mRNA in situ hybridization experiments: theendothelium of certain large vessels of the lung were stained positivewith the rabbit anti-Flt4 antiserum.

EXAMPLE 8 Identification of Flt4 Genes in Non-Human Mammalian Species

In FIG. 4 the results of an experiment examining the presence of Flt4sequences in DNA from different species is shown. In order to reveal howwell the Flt4 gene has been conserved in evolution, the 2.5 kb cDNAfragment (see FIG. 1) was hybridized to genomic DNAs purified fromdifferent animals and from yeast and digested with EcoR1. Thehybridization solution comprised 50% formamide, 5× Denhardt's solution,(100× Denhadt's solution is 2% each of Ficoll, polyvinylpyrrolidone andbovine serum albumin), 5× saline-sodium phosphate-EDTA (3M NaCl, 200 mMNaH₂PO₄—H₂O, 20 mM EDTA, pH 7.0), 0.1% sodium dodecyl sulfate, and 0.1mg/ml sonicated salmon sperm DNA. Hybridization was performed at 42° C.for 24 hours. The filter was washed at 65° C. in 1× standard salinecitrate (150 mM NaCl, 15 mM sodium citrate, pH 7.0) and 0.1% sodiumdodecyl sulfate and exposed to Kodak XAR-5 film. Specific bands werefound in monkey, rat, mouse, dog, cow, rabbit, and chick DNAs, but theyeast DNA did not give a signal. The Flt4 cDNA has been isolated fromquails. See Eichmann et al., Gene, 174(1): 3-8 (Sep. 26, 1996) andGenbank accession number X83287.

EXAMPLE 9 Flt4 Gene Expression in Adult Human Tissues

Flt4 mRNA expression in adult human tissues was analyzed using 2 μg ofpoly(A)⁺ RNA from heart, brain, placenta, lung, liver, skeletal muscle,kidney, and pancreas tissues (Multiple Tissue Northern Blot, ClontechInc.) by hybridization with the Flt4 cDNA probe. Control hybridizationswith probes for constitutively expressed genes showed an even loading ofthe lanes.

Hybridization of poly(A)⁺ RNA from various human tissues with the Flt4cDNA fragment showed mRNA bands of 5.8 and 4.5 kb mobility and a weaklylabeled band of 6.2 kb in placenta, lung, heart and kidney. Faint mRNAbands were seen in the liver and skeletal muscle, whereas the pancreasand brain appeared to contain very little if any Flt4 RNA.

EXAMPLE 10 Flt4 Expression in Human Fetal Tissues

To examine Flt4 mRNA expression in human fetal tissues, a Northern blotcontaining total RNA from the below-listed tissues of 16-19 week humanfetuses was hybridized with the 1.9 kb Flt4 cDNA fragment (see FIG. 1)and the resulting autoradiograph was scanned with a densitometer. Theresults were normalized for the amount of RNA estimated from a UVpicture of the corresponding ethidium bromide (EtBr) stained gel. Thefollowing symbols denote mRNA levels in an increasing order: −, +, ++,+++. TABLE 1 Fetal tissue mRNA Brain Meninges + Cortical plate ++Intermediate zone +++ Ependymal zone + Cerebellum ++ Choroid plexus +Liver + Pancreas + Small intestine − Heart + Lung +++ Kidney ++ Adrenal++ Skin ++ Spleen +++ Thymus −

Analysis of human fetal tissues showed that all except the thymus andsmall intestine contain Flt4 transcripts. The highest expression levelswere found in lung and spleen.

EXAMPLE 11 Flt4 Expression Vector

Full-length Flt4 cDNA (short form) was produced by a) ligation of aSphI-cleaved Flt4 PCR fragment [amplified from the S2.5 kb clone (seeFIG. 1) using the primer oligonucleotides 5′-ACATGCATGC CACCATGCAGCGGGGCGCCG CGCTGTGCCT GCGACTGTGG CTCTGCCTGG GACTCCTGGA-3′ (SEQ. ID NO.7) (forward) and 5′-ACATGCATGC CCCGCCGGT CATCC-3′ (reverse)] (SEQ. IDNO. 8) to the 5′ end of the S2.5 kb fragment, subcloned into the pSP73vector (Promega), using two SphI sites; b) ligation of a PCR fragmentcontaining the last 138 bps amplified from the 0.6 kb EcoRI fragment(see FIG. 1) with the oligonucleotide primers 5′-CGGAATTCCC CATGACCCCAAC-3′ (SEQ. ID NO. 9) (forward) and 5′-CCATCGATGG ATCCTACCTG AAGCCGCTTTCTT-3′ (SEQ. ID NO. 10) (reverse) to the 3′ end of construct a) usingthe EcoRI and BamHI sites; c) ligation of a 1.2 kb EcoRI fragment in theEcoRI site of construct b); d) ligation of the resulting full lengthHindIII-BamHI fragment into the HindIII-BamHI cleaved SV-poly expressionvector [Stacey et al., Nucl. Acids Res., 18: 2829 (1990)].

EXAMPLE 12 Identification of an Flt4 Ligand

Conditioned media from the PC-3 prostatic adenocarcinoma cell line (ATCCCRL 1435) cultured for 7 days in F12 medium in the absence of fetalbovine serum (FBS) was cleared by centrifugation at 16 000×g for 20minutes and screened for the ability to induce tyrosine phosphorylationof Flt4.

NIH3T3-cells recombinantly expressing Flt4 (see Example 13) werereseeded on 5 cm diameter cell culture dishes and grown to confluency inDulbecco's modified minimal essential medium (DMEM) containing 10% fetalbovine serum and antibiotics. The confluent cells were washed twice inphosphate-buffered saline (PBS) and starved in DMEM/0.2% bovine serumalbumin overnight. For stimulation, the starvation medium was replacedby 1 ml of the conditioned medium and the cells were incubated at 37° C.for 5 minutes.

After stimulation with the PC-3 conditioned medium, the culture platescontaining the cells were put on ice and washed twice with Tris-HCl, pH7.4, 150 mM NaCl containing 100 mM NaVO₄. The washing solution wasremoved from the dishes and the cells were lysed in RIPA buffer [10 mMTris-HCl pH 7.5, 50 mM NaCl, 0.5% sodium deoxycholate, 0.5% Nonidet P40,0.1% sodium dodecyl sulphate (SDS)] containing aprotinin, 1 mM PMSF and1 mM NaVO₄, and the lysates were sonicated for 10 seconds twice. Thelysates were then centrifuged at 16,000×g for 30 minutes and thesupernatants were transferred to new tubes and used forimmunoprecipitation.

The polyclonal antibodies against the Flt4 C-terminus (described above)were used for immunoprecipitation. Supernatants from the cell lysateswere incubated for 2 hours on ice with 2 to 4 μl of rabbit polyclonalanti-Flt4 antiserum. About 30 μl of a 50% (vol/vol) solution of proteinA-Sepharose (Pharmacia) in PBS was added, and incubation was continuedfor 45 minutes with rotation at +4° C. The immunoprecipitates werewashed three times with the RIPAo buffer and once with PBS. Theimmunoprecipitates were then subjected to SDS-polyacrylamide gelelectrophoresis (SDS-PAGE) in a 7.5% gel and blotted on nitrocellulose.These Western blots were incubated with monoclonal anti-phosphotyrosine(anti-P-Tyr) antibodies (1:2000 dilution of PT-66 Sigma, cat. P-3300)followed by detection with peroxidase-conjugated rabbit anti-mouseantibodies (1:1000 dilution, Dako, cat. P 161) using thechemiluminescence detection system (Amersham). In some cases, the blotswere stripped to clear previous signals for 30 minutes at 50° C. in 100mM 2-mercaptoethanol, 2% SDS, 62.5 mM Tris-HCl pH 6.7 with occasionalagitation and re-stained with anti-Flt4 antibodies (1:1000 dilution)followed by staining with peroxidase-conjugated swine anti-rabbitantibodies (1:1000 dilution, Dako, P217). As a positive control for thetyrosine phosphorylation of Flt4, anti-Flt4 immunoprecipitates from theFlt4-expressing NIH3T3 cells treated with 100 mM of the tyrosylphosphatase inhibitor sodium pervanadate (PerVO4) for 20 minutes wereused. Treatment of cells with Sodium pervanadate was done by addition of100 mM (final concentration) of sodium orthovanadate and 2 mM (finalconcentration) of hydrogen peroxide to the cell medium and incubation ofthe cells for 20 minutes at 37° C. 5% CO₂. That procedure resulted inthe generation of the peroxidized form of vanadate (vanadylhydroperoxide), which is a very potent inhibitor of the protein tyrosinephosphatases in living cells.

The PC-3 cell conditioned medium stimulated tyrosine phosphorylation ofa 120 kD polypeptide which co-migrated with tyrosine phosphorylated,processed mature form of Flt4. Co-migration was confirmed afterrestaining of the blot with anti-Flt4 antibodies.

To prove that 120 kD polypeptide is not a non-specific component of theconditioned medium, 15 ml of conditioned medium were separated bySDS-PAGE, blotted on nitrocellulose, and the blot was stained withanti-P-Tyr antibodies. Several polypeptides were detected, but none ofthem comigrated with Flt4, indicating that the 120 kD band is indeedtyrosine-phosphorylated protein immunoprecipitated from the stimulatedcells. Analysis of stimulation by PC-3 conditioned medium pretreatedwith heparin Sepharose CL-6B (Pharmacia) for 2 hours at room temperature(lane 3) shows that the Flt4 ligand does not bind to heparin.

Unconditioned medium did not induce Flt4 autophosphorylation. Also,neither non-transfected NIH3T3 cells nor NIH3T3 cells transfected withthe FGFR-4 showed tyrosine phosphorylation of the 120 kD polypeptideupon stimulation with the conditioned medium from PC-3 cells.Stimulating activity was considerably increased when the PC-3conditioned medium was concentrated fourfold using a Centricon-10concentrator (Amicon). Also, the flow through obtained after theconcentration, containing proteins of less than 10,000 molecular weight(<10,000), did not stimulate phosphorylation of Flt4. Pretreatment ofthe concentrated conditioned medium of PC-3 cells with 50 ml of the Flt4extracellular domain (Flt4EC-6×His, see below) coupled to CNBr-activatedSepharose (1 mg/ml) according to the manufacturer's instructionscompletely abolished the tyrosine phosphorylation of Flt4. Analogouspretreatment of the conditioned medium with Sepharose CL-4B did notaffect its stimulatory activity.

These data prove that PC-3 cells produce soluble ligand for Flt4. Theabove experiments prove that the ligand binds to the recombinant Flt4 ECdomain. Thus, that ligand can be purified using the recombinant Flt4 ECdomain in affinity chromatography. The purified protein can beelectrophoresed in SDS-PAGE, blotted onto polyvinylidene difluoride(PVDF) membranes and its amino terminal sequence can be determined bymethods standard in the art. Alternatively, the purified ligand can bedigested to peptides for their amino terminal sequence determination.Peptide sequences obtained from the purified protein are used for thesynthesis of a mixture of oligonucleotides encoding such sequences. Sucholigonucleotides and their complementary DNA strand counterparts can beradioactively labelled by and used for the screening of cDNA librariesmade from the PC-3 cells to obtain a cDNA encoding the ligand, all bymethods standard in the art (Wen et al., Cell 69: 559-572 (1992)).Alternatively, such oligonucleotides and their counterparts can be usedas primers in polymerase chain reaction (PCR) to amplify sequencesencoding the ligand using cDNA made from PC-3 cell RNA as a template.Such method of cDNA synthesis and PCR (RT-PCR) is standard in the art(Innis et al., 1990, PCR protocols, Academic Press; McPherson, M. J. etal., 1991, PCR, a practical approach, IRL Press; Partanen et al., Proc.Natl. Acad. Sci., USA, 87: 8913-8917 (1990)). Yet another alternative isto clone the Flt4 ligand from the PC-3 cells by using cDNAs cloned intoeukaryotic expression vector (e.g. using the Invitrogen Librariancloning kit and vectors provided, such as pcDNA I or pcDNA III) andscreening of such libraries transfected into, e.g., COS cells withFlt4-alkaline phosphatase (Cheng and Flanagan, Cell, 79: 157-168,(1994)), Flt4-immunoglobulin (Flt4-Ig) (Lyman et al., Cell, 75:1157-1167 (1993)), or similar affinity reagents, by methods standard inthe art.

EXAMPLE 13 Cell Lines and Transfections

NIH3T3 cells and 293-EBNA cells (Invitrogen) were cultured in DMEMcontaining 10% FCS. For stable expression, NIH3T3 cells were transfectedwith the LTR-FLT41 vector together with the pSV-neo vector (see Example4, above) where the Flt4 cDNA is expressed under the control of theMoloney murine leukemia virus LTR promoter, by the lipofection methodusing the DOTAP transfection reagent (Boehringer-Mannheim). COS-1 cellswere transfected by the DEAE dextran method (McClutchan and Pagano, J.Natl. Cancer Inst., 41: 351-35 (1968)). Transfected cells were selectedin 500 mg/ml neomycin.

EXAMPLE 14 Construction and Expression of Flt4 Fusion Proteins

The pVTBac-FLT4EC-6×His fusion construct. The ends of cDNA encoding Flt4were modified as follows: The 3′ end of Flt4 cDNA sequence encoding theextracellular domain (EC) was amplified using oligonucleotides5′-CTGGAGTCGACTTGGCGGACT-3′ (SEQ ID NO: 13, SalI site underlined,containing sequence corresponding to nucleotides 2184-2204 of SEQ IDNO: 1) and 5′CGCGGATCCCTAGTGATGGTGATGGTGATGTCTACCTTCGATCATGCTGCCCTTATCCTC-3′ (SEQ ID NO: 14, BamHI siteunderlined, containing sequence complementary to nucleotides 2341-2324of SEQ ID NO: 1) encoding 6 histidine residues for binding to a Ni-NTAcolumn (Qiagen, Hilden, Germany) followed by a stop codon. The amplifiedfragment was digested with SalI and BamiHI and ligated as a SalI-BamHIfragment into the LTR-FLT41 vector (see Example 4), replacing a uniqueSalI-BamHI fragment containing sequences encoding the Flt4 transmembraneand cytoplasmic domains.

The 5′ end of the Flt4 cDNA without the Flt4 signal sequence encodingregion was amplified by PCR using oligonucleotides5′-CCCAAGCTTGGATCCAAGTGGCTACTCCATGACC-3′ (SEQ ID NO: 11, HindIII andBamHI sites underlined, containing sequence corresponding to nucleotides86-103 of SEQ ID NO: 1) and 5′-GTTGCCTGTGATGTGCACCA-3′ (SEQ ID NO: 12,containing sequence complementary to nucleotides 700-681 of SEQ ID NO:1). This amplified fragment (which included nucleotides 86-700 of SEQ IDNO: 1) was digested with HindIII and SphI (the SphI site, correspondingto nucleotides 588-593 of SEQ ID NO: 1, being within the amplifiedregion of the Flt4 cDNA).

The resultant HindIII-SphI fragment was used to replace a HindIII-SphIfragment in the modified LTR-FLT41 vector described immediately above(the HindIII site is in the 5′ junction of the Flt4 insert with thepLTRpoly portion of the vector, the SphI site is in the Flt4 cDNA andcorresponds to nucleotides 588-593 of SEQ ID NO: 1). The resultantFIt4EC-6×His insert was then ligated as a BamHI fragment into the BamHIsite in the pVTBac plasmid (Tessier et al., Gene 98: 177-183(1991)). Theconstruct was transfected together with baculovirus genomic DNA intoSF-9 cells by lipofection. Recombinant virus was generated and used forinfection of High-Five cells (Invitrogen).

The Flt4-AP fusion construct. The 3′ end of the sequence encoding theFlt4 EC domain was amplified using oligonucleotides5′-CTGGAGTCGACTTGGCGGACT-3′ (SEQ ID NO: 15) and5′-CGGGATCCCTCCATGCTGCCCTTATCCT-3′ (SEQ ID NO: 16) and ligated asSalI-BamHI fragment into the LTR-FLT41 vector, replacing sequencesencoding the transmembrane and cytoplasmic domains. The resulting insertwas then ligated as a HindIII-BamHI fragment into the HindIII-BglIIsites of plasmid APtag-1 in frame with the alkaline phosphatase codingregion (Flanagan and Leder, 1990, Cell 63, 185-194). NIH3T3 cells wereco-transfected with this Flt4-AP construct and pSV2neo (Southern andBerg, J. Mol. Appl. Genet. 1: 327-341 (1982)) by lipofection using theDOTAP transfection reagent (Boehringer) and the transfected cells wereselected in the presence of 500 mg/ml neomycin. The recombinant proteinproduced into the medium was detected by a colorimetric reaction forstaining for alkaline phosphatase activity (Cheng and Flanagan, Cell 79:157-168 (1994)).

The Flt4-Ig construct. A recombinant DNA encoding an Flt4-immunoglobulinchimera was constructed as follows. The 5′ end of the cDNA encodingFlt4, including Flt4 nucleotides encoding the signal sequence, wasamplified by PCR using primers5′-GGCAAGCTTGAATTCGCCACCATGCAGCGGGGCGCC-3′ (SEQ ID NO: 17) and5′-GTTGCCTGTGATGTGCACCA-3′ (SEQ ID NO: 18) and ligated as HindIII-SphIfragment into the LTR-FLT41 vector. The 3′ end of Flt4 EC-encodingsequence was amplified using oligonucleotides5′-CTGGAGTCGACTTGGCGGACT-3′ (SEQ ID NO: 19) and5′-CGCGGATCCAAGCTTACTTACCTTCCATGCTGCCCTTATCCTCG-3′ (SEQ ID NO: 20) andligated as SalI-BamHI fragment into the LTR-FLT41 vector replacing thesequences encoding the transmembrane and cytoplasmic domains. ThisFlt4EC insert containing a splice donor site was ligated first intopHγCE2 containing exons encoding the human immunoglobulin heavy chainhinge and constant region exons (Karjalainen, K., TIBTECH, 9: 109-113(1991)). The EcoRI-BamHI insert containing the Flt4-Ig chimera was thenblunted by methods standard in the art (Klenow) and ligated to theblunted HindIII site in pREP7 (Invitrogen). The construct wastransfected into 293-EBNA cells by the calcium-phosphate precipitationmethod and the conditioned medium was used for the isolation of theFlt4-Ig protein by protein A-Sepharose affinity chromatography.

EXAMPLES 15-17 Purification and Sequencing the Flt4 Ligand

Cell culture supernatants produced by PC-3 cells under serum-depletedconditions are concentrated 30-50 fold using Centriprep filtercartridges and loaded onto a column of immobilized Flt4 extracellulardomain. Two affinity matrices are prepared using the alternativeconstructs and methods. In the first case the Flt4EC-6×His fusionprotein is crosslinked to CNBr-activated Sepharose 4B (Pharmacia) and inthe second case the Flt4-Ig fusion protein is coupled to protein ASepharose using dimethylpimelidate (Schneider et al., 1982, J. Biol.Chem. 257: 10766-10769). The material eluted from the affinity column issubjected to further purification using ion exchange and reverse-phasehigh pressure chromatography and SDS-polyacrylamide gel electrophoresis.Chromatography fractions are tested for the ability to stimulatetyrosine phosphorylation of Flt4. The purified biologically activeligand protein is microsequenced and the degenerate oligonucleotides aremade based on the amino acid sequence obtained, for the purpose ofisolating and cloning a ligand-encoding cDNA; e.g., from a cDNA librarygenerated from poly(A)⁺ RNA isolated from PC-3 cells.

A detailed characterization of an Flt4 ligand, designated VascularEndothelial Growth Factor C (VEGF-C), as well as native human, non-humanmammalian, and avian polynucleotide sequences encoding VEGF-C, andVEGF-C variants and analogs, is provided in International PatentApplication Number PCT/US98/01973, filed 02 Feb. 1998 (published 06 Aug.1998 as International Publication Number WO 98/33917); in Joukov et al.,J. Biol. Chem., 273(12): 6599-6602 (1998); in Joukov et al., EMBO J,16(13): 3898-3911 (1997); and in International Patent Application No.PCT/FI96/00427, filed Aug. 1, 1996 (published as InternationalPublication No. WO 97/05250), all of which are incorporated herein byreference in the entirety. As explained therein in detail, human VEGF-Cis initially produced in human cells as a prepro-VEGF-C polypeptide of419 amino acids. An amino acid sequence for human prepro-VEGF-C is setforth in SEQ ID NO: 21, and a cDNA encoding human VEGF-C has beendeposited with the American Type Culture Collection (ATCC), 10801University Blvd., Manassas, Va. 20110-2209 (USA), pursuant to theprovisions of the Budapest Treaty (Deposit date of 24 Jul. 1995 and ATCCAccession Number 97231). VEGF-C sequences from other species also havebeen reported. See Genbank Accession Nos. MMU73620 (Mus musculus); andCCY15837 (Coturnix coturnix) for example, incorporated herein byreference.

The prepro-VEGF-C polypeptide is processed in multiple stages to producea mature and most active VEGF-C polypeptide of about 21-23 kD (asassessed by SDS-PAGE under reducing conditions). Such processingincludes cleavage of a signal peptide (SEQ ID NO: 21, residues 1-31);cleavage of a carboxyl-terminal peptide (corresponding approximately toamino acids 228-419 of SEQ ID NO: 21 and having a pattern of spacedcysteine residues reminiscent of a Balbiani ring 3 protein (BR3P)sequence [Dignam et al., Gene, 88:133-40 (1990); Paulsson et al., J.Mol. Biol., 211:331-49 (1990)]) to produce a partially-processed form ofabout 29 kD; and cleavage (apparently extracellularly) of anamino-terminal peptide (corresponding approximately to amino acids32-103 of SEQ ID NO: 21) to produced a fully-processed mature form ofabout 21-23 kD. Experimental evidence demonstrates thatpartially-processed forms of VEGF-C (e.g., the 29 kD form) are able tobind the Flt4 (VEGFR-3) receptor, whereas high affinity binding toVEGFR-2 occurs only with the fully processed forms of VEGF-C. It appearsthat VEGF-C polypeptides naturally associate as non-disulfide linkeddimers.

Moreover, it has been demonstrated that amino acids 103-227 of SEQ IDNO: 2 are not all critical for maintaining VEGF-C functions. Apolypeptide consisting of amino acids 113-213 (and lacking residues103-112 and 214-227) of SEQ ID NO: 2 retains the ability to bind andstimulate VEGF-C receptors, and it is expected that a polypeptidespanning from about residue 131 to about residue 211 will retain VEGF-Cbiological activity. The cysteine residue at position 156 has been shownto be important for VEGFR-2 binding ability. However, VEGF-C ΔC₁₅₆polypeptides (i.e., analogs that lack this cysteine due to deletion orsubstitution) remain potent activators of VEGFR-3. The cysteine atposition 165 of SEQ ID NO: 2 is essential for binding either receptor,whereas analogs lacking the cysteines at positions 83 or 137 competewith native VEGF-C for binding with both receptors and stimulate bothreceptors.

An alignment of human VEGF-C with VEGF-C from other species (performedusing any generally accepted alignment algorithm) suggests additionalresidues wherein modifications can be introduced (e.g., insertions,substitutions, and/or deletions) without destroying VEGF-C biologicalactivity. Any position at which aligned VEGF-C polypeptides of two ormore species have different amino acids, especially different aminoacids with side chains of different chemical character, is a likelyposition susceptible to modification without concomitant elimination offunction. An exemplary alignment of human, murine, and quail VEGF-C isset forth in FIG. 5 of PCT/US98/01973.

Apart from the foregoing considerations, it will be understood thatinnumerable conservative amino acid substitutions can be performed to awildtype VEGF-C sequence which are likely to result in a polypeptidethat retains VEGF-C biological activities, especially if the number ofsuch substitutions is small. By “conservative amino acid substitution”is meant substitution of an amino acid with an amino acid having a sidechain of a similar chemical character. Similar amino acids for makingconservative substitutions include those having an acidic side chain(glutamic acid, aspartic acid); a basic side chain (arginine, lysine,histidine); a polar amide side chain (glutamine; asparagine); ahydrophobic, aliphatic side chain (leucine, isoleucine, valine, alanine,glycine); an aromatic side chain (phenylalanine, tryptophan, tyrosine);a small side chain (glycine, alanine, serine, threonine, methionine); oran aliphatic hydroxyl side chain (serine, threonine). Addition ordeletion of one or a few internal amino acids without destroying VEGF-Cbiological activities also is contemplated.

From the foregoing, it will be appreciated that many VEGF-C polypeptidesand variants will bind Flt4 (VEGFR-3) with high affinity and thereforeare useful as Flt4 binding compounds in aspects of the invention thatinvolve imaging or screening of tissue samples using a Flt4 bindingcompound. Of particular interest are forms of VEGF-C harboringalterations which diminish or eliminate VEGFR-2 binding affinity, suchthat the resultant polypeptide possesses increased binding specificityfor VEGFR-3. As described above, such alterations include the deletionor replacement of Cys₁₅₆, which substantially eliminates VEGFR-3 bindingaffinity, or amino acid sequence alterations that destroy naturalprepro-VEGF-C proteolytic processing sites (since VEGFR-2 affinity ishighest with fully processed VEGF-C). In addition, VEGF-C molecules thathave been modified to retain Flt4 binding affinity but that fail toactivate Flt4 autophosphorylation are useful Flt4 antagonists in methodsof treatment described herein. It will further be apparent from theforegoing teachings that the Flt4 ligand described herein may be used inassays as an additional indicia to confirm the identity of human Flt4allelic variants, and to confirm that non-human gene sequences havinghomology to the Flt4 sequences taught herein (See, e.g., Example 8 andFIG. 4) are in fact the non-human counterparts to Flt4. The deducedamino acid sequence for prepro-VEGF-C is set forth herein in SEQ ID NO:21.

A detailed description of a second Flt4 ligand, designated VascularEndothelial Growth Factor D (VEGF-D), as well as human polynucleotidesequences encoding VEGF-D, and VEGF-D variants and analogs, is providedin International Patent Application Number PCT/US97/14696, filed 21 Aug.1997 and published on 26 Feb. 1998 as International Publication NumberWO 98/07832; and Achen, et al., Proc. Nat'l Acad. Sci. U.S.A., 95(2):548-553 (1998), also incorporated herein by reference. As explainedtherein in detail, human VEGF-D is initially produced in human cells asa prepro-VEGF-D polypeptide of 354 amino acids. The cDNA and deducedamino acid sequences for prepro-VEGF-D are set forth herein in SEQ IDNO: 22. VEGF-D sequences from other species also have been reported. SeeGenbank Accession Nos. D89628 (Mus musculus); and AF014827 (Rattusnorvegicus), for example, incorporated herein by reference.

The prepro-VEGF-D polypeptide has a putative signal peptide of 21 aminoacids and is apparently proteolytically processed in a manner analogousto the processing of prepro-VEGF-C. A “recombinantly matured” VEGF-Dlacking residues 1-92 and 202-354 of SEQ ID NO: 22 retains the abilityto activate receptors VEGFR-2 and VEGFR-3, and appears to associate asnon-covalently linked dimers. The utilities for VEGF-D polypeptides asFlt4 binding compounds in the invention are analogous to those describedabove for VEGF-C. Likewise, it is expected that analogous alterations toVEGF-D (to eliminate the second of eight conserved cysteines in the VEGFhomology domain, Cys₁₃₆, or to eliminate proteolytic processing sites)will result in polypeptides having reduced or eliminated VEGFR-2 bindingaffinity and, thus, increased Flt4 specificity. VEGF-D molecules thathave been modified to retain Flt4 binding affinity but that fail toactivate Flt4 autophosphorylation are useful Flt4 antagonists in methodsof treatment described herein.

EXAMPLE 18 Cloning of Mouse Flt4 cDNA Probes

Approximately 10⁶ plaques from a λFI®II genomic library from 129SV mice(Stratagene) was screened with the S2.5 human Flt4 receptor cDNAfragment described above, covering the extracellular domain. See alsoPajusola et al., Cancer Res., 52:5738 (1992). A 2.5 kb BamHI fragmentwas subcloned from a positive plaque and sequenced from both ends. Fromthis subclone, polymerase chain reaction was used to amplify and cloneinto the pBluescript KSII+/− vector (Stratagene) an exon fragmentcovering nucleotides 1745-2049 of the mouse Flt4 cDNA sequence. SeeFinnerty et al., Oncogeze, 8:2293 (1993).

A second fragment covering nucleotides 1-192 was similarly cloned.

EXAMPLE 19 Analysis of Flt4 mRNA in Mouse Tissues

Total RNA was isolated from developing embryos (8-18 days p.c. and oneday old mice) according to Chomczynski et al., Anal. Biochem., 162:156(1987). The sample from 8 day p.c. embryos also included the placenta.

For RNase protection analysis, RNA probe was generated from thelinearized murine Flt4 plasmid obtained according to Example 18 using[³²P]-UTP and T7 polymerase for the antisense orientation. The β-actinprobe used corresponds to nucleotides 1188-1279 of the published mouseβ-actin sequence. See Tokunaga, et al., Nucleic. Acid. Res., 14:2829(1986). After purification in a 6% polyacrylamide/7M urea gel, thelabelled transcripts were hybridized to 30 μg of total RNA overnight at52° C. Unhybridized RNA was digested with RNase A (10 U/ml) and T1 (1mg/ml) at 37° C., pH 7.5 for 1 hour. The RNases were inactivated byproteinase K digestion at 37° C. for 15 minutes and the samples wereanalysed in a 6% polyacrylamide/7M urea gel.

The pattern of expression of Flt4 analysed in this experiment showedthat very weak mRNA signals were obtained from lung, liver, heart,kidney, skeletal muscle and spleen, whereas testis and brain wereapparently without specific signal. Analysis of a series of RNAscollected during different phases of mouse development by RNaseprotection assay showed that the Flt4 mRNA was expressed throughoutembryogenesis from day 8 p.c. to newborn mice without great variationsin signal intensity.

EXAMPLE 20 In Situ Hybridization for Flt4 in Mouse Embryos

To better assign Flt4 transcripts to cells and tissues, sections of 7.5and 8.5 day p.c. mouse embryos were hybridized with labelled Flt4 RNAs.Mouse embryos were derived from matings of CBA and NMRI mice. Pregnantmice were killed by cervical dislocation and the embryos were eitherimmediately frozen or transferred via phosphate buffered saline into 4%paraformaldehyde. The embryos and isolated mouse organs were fixed for18 hours at 4° C., dehydrated, embedded in paraffin, and cut into 6 μmsections.

RNA probes (antisense and sense) of 192 and 305 nucleotides (see Example18) were generated from linearized plasmids using [³⁵S]-UTP. In situhybridization of sections was performed according to Wilkinson et al.,Development, 99:493 (1987); and Wilkinson et al., Cell, 50:79 (1987),incorporated by reference herein, with the following modifications:1)-instead of toluene, xylene was used before embedding in paraffin wax;2) 6 μm sections were cut, placed on a layer of diethylpyrocarbonate-treated water on the surface of glass slides pretreatedwith 2% 3-triethoxysilylpropylamine; 3) alkaline hydrolysis of theprobes was omitted; and 4) the high stringency wash was for 80 minutesat 65° C. in a solution containing 30 mM DTT and 1×SSC. The sectionswere covered with NTB-2 emulsion (Kodak) and stored at 4° C. The slideswere exposed for 14 days, developed, and stained with hematoxylin.Control hybridizations with sense strand and RNase A-treated sectionsdid not give a specific signal above background.

Flt4 mRNA expression was not detected in 7.5 day p.c. mouse embryos, butbright signals were detected in the developing aortae on day 8.5 ofdevelopment. In contrast, the developing yolk sac was Flt4-negative. Inthe extraembryonic tissues, Flt4 was prominently expressed in theallantois, whereas developing blood islands of the yolk sac werenegative. On the other hand, angioblasts of the head mesenchyme werestrongly Flt4-positive. In the developing placenta, Flt4 signal wasfirst seen in peripheral sinusoidal veins. In 9.5 day p.c. placenta, theendothelium of venous lacunae and the giant cells partially fused to theReichert's membrane expressed Flt4 mRNA.

Thus, although Flt4 expression was very prominent in the earliestendothelial cell precursors, the angioblasts, it appeared to berestricted only to certain vessels of 8.5 day p.c. embryos. The Tiereceptor is known to be expressed in all endothelial cells of developingmouse embryos and thus provides a marker for these cells. See Korhonen,et al. Oncogene, 8:395 (1993); and Korhonen et al., Blood, 80: 2548-2555(1992). Notably, in contrast to the Tie probe, the Flt4 probe hybridizedvery weakly if at all with arterial endothelia of 11.5 day p.c. embryos,e.g. with the endothelium of the developing dorsal aorta or the carotidarteries. Instead, Flt4 signal was much more prominent in the developingveins. For example, Flt4 signal was detected in veins surrounding thedeveloping metanephros, while the Tie probe predominantly recognizedcapillaries within the metanephros.

Flt4 mRNA was observed to be distributed in several regions of a 12.5day p.c. mouse embryo, being particularly prominent in the dilatedvessel of the axillar region. A similar Flt4-positive vessel structurewas seen in the mid-sagittal section in the jugular area (data notshown). A plexus-like pattern of Flt4-expressing vessels appeared in theperiorbital region and surrounding the developing vertebrae. Also, justbeneath the developing skin, a Flt4-positive vascular network wasevident. Weaker capillary signals were obtained from several regions,including the developing brain. Flt4 mRNA could also be detected insmall vessels of the neck region, of the developing snout and at thebase of the developing tongue as well as in the tail region.Additionally, the liver was strongly positive for Flt4 mRNA in aspotlike pattern.

During further development, Flt4 mRNA appeared to become more restrictedto certain vessels of the embryo. A 14.5 day p.c. embryo shows nicelythis restricted pattern of expression. In the midsagittal section fromsuch an embryo, the most prominent Flt4 signal was observed along, thedeveloping vertebral column in its anterior part. This signal wasconsidered to originate from endothelial cells of the thoracic duct,which is the largest lymphatic vessel formed at this time ofdevelopment. In contrast, the dorsal aorta and inferior vena cava werenegative. Dilated vessels in the mesenteric region were also stronglypositive for Flt4. Furthermore, as in the 12.5 day p.c. embryos, vesselnetworks along anatomical boundaries in the periorbital, lower jaw, aswell as in the neck regions contained Flt4-positive endothelia. Similarstuctures were present in the pericardial space and throughout thesubcutaneous tissue. Notably, in contrast to Flt4-negative vessels, allFlt4-positive vessels were devoid of blood cells in their lumen. Theseexpression patterns suggest that Flt4 becomes confined to the endotheliaof lymphatic vessels at this time of development. An additional sitewhere we observed Flt4 expression was in the sinusoids of the developingbone marrow.

A transverse section of the upper thorax of a 16.5 day p.c. embryohybridized with the Flt4 probe also was analyzed. Hematoxylin-eosinstaining was performed to visualize the different types of vessels inthis area. These include the carotid and brachiochepalic arteries, thevena cava, and the thoracic duct, which is smaller in size and lackssurrounding muscular and connective tissue. Under higher magnificationendothelial cells of the thoracic duct as well as a small vessel in thevicinity were observed to hybridize with the Flt4 probe.

EXAMPLE 21 Analysis of Flt4 mRNA in Cultured Endothelial Cells

The in situ hybridization results described in Example 20 showed thatFlt4 is expressed in venous endothelial cells and later in lymphaticvessels and some venous endothelial cells, but not in arterialendothelia. In order to determine if such regulation was maintained invitro, we studied cultured endothelial cells using Northern blotting andhybridization analysis.

Endothelial cells from human aorta, femoral vein, umbilical vein, andfrom foreskin microvessels were isolated, cultured, and characterized aspreviously described in the art. See Van Hinsberg et al.,Arteriosclerosis, 7:389 (1987); and Van Hinsberg, et al., Thromb.Haemostas, 57:148 (1987). They were used at confluent density after fiveto eight passages (split ratio 1:3) for the isolation of polyadenylatedRNA.

The endothelial cell lines EA hy926 (Edgell et al., Proc. Natl. Acad.Sci., 80: 3734-3737 (1983)), BCE (Folkman et al., Proc. Natl. Acad.Sci., 76: 5217-5221 (1979)) and LEII (Schreiber et al., Proc. Natl.Acad. Sci., 82: 6138 (1985)) did not express Flt4. However, culturedhuman microvascular, venous and umbilical vein endothelial cells werepositive for the Flt4-specific 5.8 and 4.5 kb mRNAs, whereas the aorticendothelial cells were negative. In contrast, another endothelialreceptor tyrosine kinase gene, tie, was expressed as a 4.4 kb mRNA inall endothelial cell types studied.

EXAMPLE 22 Flt4 mRNA in in Adult Human Tissues

The results obtained in Example 20 indicated that the Flt4 mRNA becomeslargely confined to the endothelium of lymphatic vessels duringdevelopment. Because of the potential significance of this finding inhumans, we also studied Flt4 expression in adult human tissues using ahuman Flt4 probe. The human Flt4 probe used was an EcoRI-SphI fragmentcovering base pairs 1-595 of the cDNA (SEQ ID NO:1). See also Pajusolaet al., Cancer Res., 52:5738 (1992). The von Willebrand factor probe wasan EcoRI-HindIII fragment covering base pairs 1-2334. Bonthron, et al.,Nucleic Acids Res., 141:7125 (1986).

We used routinely fixed material sent for histopathological diagnosis.Normal lung tissue was obtained from a resection of the left inferiorlung lobe affected by epidermoid cancer. Mesenterium and mesenteriallymph nodes were obtained from a patient having a colonicadenocarcinoma. A normal lymph node adjacent to the salivary gland wasenucleated because of its abnormal size. The tonsils from two patientsand the two appendixes had no diagnostic changes. Two lymphangiomyoniasand three cystic lymphangiomas were studied with similar results.

For human tissues, which were routine samples fixed with 10% formalinfor histopathological diagnosis, the normal in situ protocol gave justbackround, whereas microwave treatment instead of proteinaase K enabledspecific hybridization. Shi, et al., J. Biol. Chem. 266:5774 (1991);Catoretti, et al., J. of Pathol., 168:357 (1992).

In the mesenterium, lung and appendix lymphatic endothelia gave Flt4signals, while veins, arteries, and capillaries were negative. To studywhether Flt4 is expressed in the HEVs, the tonsils were studied. Indeed,in the tonsils, Flt4-specific autoradiographic grains were detected insome HEVs.

EXAMPLE 23 Analysis of Flt4 mRNA in Normal and Metastatic Lymph Node andin Lymphangioma

A portion of a human mesenterial lymph node (see Example 22) wasanalysed for Flt4 expression. Flt4 expression was observed in thelymphatic sinuses and afferent and efferent lymphatic vessels. The samepattern was observed in a lymph node containing adenocarcinomametastases. Some HEVs in both normal and metastatic lymph node were alsopositive. Flt4 expression in a cystic lymphangioma was specific tolymphatic endothelia, as evident from a comparison with the in situsignals for von Willebrandt factor in all blood vessels.

Consistent with these results, immunostaining for Flt4 was stronglypositive in the endothelium of cutaneous lymphangiomatosis, a raredisorder characterized by proliferation of presumed lymphaticendothelium. See Lymboussaki et al., Am. J. Pathol., 153(2): 395-403(August, 1998), incorporated herein by reference in its entirety.

Additionally, immunostaining for Flt4 identified spindle cells withinKaposi's sarcoma cutaneous nodular lesion tissue samples. See Jussila etal., Cancer Res., 58:1599-1604 (April, 1998). In view of the apparentlymphatic specificity of Flt4, These results may be consideredconsistent with suggestions that cerain cells in Kaposi's sarcoma are oflymphatic endothelial origin. See, e.g., Beckstead et al. Am J. Pathol.,119: 294-300 (1985); and Dictor et al., Am J. Pathol., 130: 411-417(1988).

EXAMPLE 24 Localization of Flt4 in Fetal Endothelial Cells

As described in Example 2, An Flt4 cDNA fragment encoding the 40 carboxyterminal amino acids of the short form was cloned as a 657 bpEcoRI-fragment into the pGEX-1λT bacterial expression vector (Pharmacia)in-frame with the glutatione-S-transferase coding region. The resultantGST-Flt4 fusion protein was produced in E. coli and purified by affinitychromatography using a glutathione-Sepharose 4B column. The purifiedprotein was lyophilized, disolved in PBS, mixed with Freund's adjuvant,and used for immunization of rabbits. Antisera were used after the thirdbooster immunization.

Tissues from 17 and 20-week-old human fetuses were obtained from legalabortions induced with prostaglandins. The study was approved by theEthical Committee of the Helsinki University Central Hospital. Thegestational age was estimated from the fetal foot length. The fetaltissues were embedded in Tissue-Tek (Miles), frozen immediately, andstored at −70° C.

Anti-Flt4 antiserum was cross-absorbed to a GST-Sepharose column toremove anti-GST-antibodies and then purified by GST-Flt4 affinitychromatography. Several 6 μm-thick cryostat sections of the tissues werefixed with acetone and treated with 0.3% H₂O₂ in methanol for 30 minutesto block endogenous peroxidase activity. After washing, the sectionswere incubated with 5% normal swine serum. Sections were then incubatedwith antibodies against Flt4 and washed. Bound antibodies were detectedwith peroxidase-conjugated swine anti-rabbit IgG followed by stainingfor peroxidase activity using 0.2% 3,3-diaminobenzidine (Amersham) as asubstrate. The sections were counterstained in Meyer's hematoxylin.

Anti-Flt4 immunoperoxidase staining of human fetal mesenterium showedFlt4 protein in the endothelium of several vessels, while controlstainings with antigen-blocked anti-Flt4 antibodies and preimmune serawere negative. For comparison, sections were stained with an antiserumagainst the Factor VIII-related antigen, which is specific for vascularendothelial cells. Immunoperoxidase staining for Flt4 was observed overendothelial cells of vessels, which did not contain red blood cells,while blood vessels were negative. The vessels without red blood cellsare likely to be lymphatic endothelial cells; such vessels areparticularly frequent in the mesenterium. The antibodies against FactorVIII related antigen stained endothelial cells in all vessels.

EXAMPLE 25 Production of Monoclonal Antibodies Against Flt4

Fusion I:

Recombinant Flt4 extracellular domain protein was produced by expressingthe Flt4EC-6×His-pVTBac plasmid construct (Example 14) in High-Fivecells. The Flt4 extracellular domain (Flt4EC) was purified from theculture medium of the infected High-Five cells using Ni-NTA affinitychromatography according to manufacturer's instructions (Qiagen) forbinding and elution of the 6×His tag encoded in the COOH-terminus of therecombinant Flt4 extracellular domain.

Four month old Balb/c male mice were immunized by intraperitonealinjection of the purified, recombinantly produced Flt4 extracellulardomain protein (150 μg/mouse) emulsified with Freund's completeadjuvant. Booster injections of 150 μg were given at three to four weekintervals and a final booster (10 μg Flt4 EC in PBS, administeredintraperitoneally) was given after another three-week interval. Fourdays after the final booster dose, the mice were sacrificed and mousesplenic lymphoid cells were fused with SP 2/0 plasmacytoma cells at a2:1 ratio, respectively.

The fused cells were harvested in 96-well culture plates (NUNC) inEx-Cell 320 medium (SERALAB) containing 20% fetal calf serum and HATsupplement (hypoxanthine-aminopterin-thymidine; GIBCO, 043-01060H;diluted 50-fold). Cells were cultured at +37° C., in a 5% CO₂atmosphere. After 10 days, HAT-supplemented medium was changed toHT-supplemented cell culture medium (GIBCO; 043-01065H, diluted50-fold). HT medium is identical to HAT medium, but lacks aminopterin.

In three weeks, specific antibody production was determined by theantigen-specific ImmunoFluoroMetric Assay, (IFMA), described below inExample 26. The master clones were cloned by limited dilutions asdescribed by Staszewski et al., Yale Journal of Biology and Medicine,57:865-868 (1984). Positive clones were expanded onto 24-well tissueculture plates (NUNC), recloned, and re-tested by the same method.Positive clones were tested by fluorescence-activated cell sorting(FACS).

The stable clones secreted immunoglobulins belonging to the IgG, class,except one, which produced Ig probably belonging to class IgA. Thesubclass of monoclonal antibody was determined using rat monoclonalantibody to mouse subclass as biotin conjugate (SEROTEC) in IFMA.

Balb/c mice were used to produce monoclonal antibodies in ascites fluid.The hybridomas described above were intraperitoneally injected into miceafter pretreatment of the animals with pristane(2,6,10,14-tetramethylpentadecan 98%, ALDRICH-CHEMIE D7924 Steinheim,Cat. No. T 2,280-2). 0.5 ml of pristane (i.v.) was injected about twoweeks prior to the hybridoma cells. The amount of cells injected wereapproximately 7.5 to 9×10⁶ per mouse. Ascites was collected 10 to 14days after injection of the hybridomas.

Fusion II:

Two month old Balb/c mice (female) were immunized by intraperitonealinjection of the recombinantly produced Flt4 extracellular domainprotein (20 μg/mouse), emulsified with Freund's complete adjuvant.Booster injections of 20 μg were given at three to four week intervalsand a final booster (10 μg Flt4 in PBS, administered i.v.) was givenafter another three-week interval. Four days after the final boosterdose, the mice were sacrificed and mouse splenic lymphoid cells werefused with SP 2/0 plasmacytoma cells at a 2:1 ratio, respectively.

The fused cells were harvested in 96-well culture plates (FALCON) inOptiMEM 1 (with Glutamax, 1, 51985-026, GIBCO BRL) medium containing 20%fetal calf serum and HAT supplement (hypoxanthine-aminopterin-thymidine,GIBCO BRL 21060-017; diluted 1:50 fold). Cells were cultured at 37° C.,in a 5% CO₂ atmosphere. After 10 days, HAT-supplemented medium waschanged to HT-supplemental cell culture medium (GIBCO BRL; 41065-012,diluted 1:50-fold).

In three weeks, specific antibody production was determined by theantigen-specific ImmunoFluoroMetric Assay (IFMA) described below inExample 26. The master clones were cloned by limited dilutions asdescribed by Staszewki et al. (1984). Positive clones were expanded onto24-well tissue culture plates (FALCON), re-cloned, and re-tested by thesame method. Positive clones were tested by FACS.

The 2E11 and 6B2 clones secreted immunoglobulins belonging to the IgG₁class, and 2B12 clones produced Ig belonging to subclass IgM. The mousesubclass IgG₁ was determined using rat monoclonal antibody against mousesubclass heavy chain as biotin conjugate (SEROTEC) in IFMA and the mousesubclass IgM was determined with Mouse Monoclonal Antibody Isotyping Kit(Dipstick Format) (19663-012, Life Technologies Inc.).

EXAMPLE 26 Specificity of Monoclonal Antibodies Against Flt4

The purified, recombinant Flt4 extracellular domain-6×His fusion product(produced as described in Examples 14 and 25) was labelled with Europiumaccording to Mukkala et al., Anal. Biochem, 176(2):319-325 (1989), withthe following modification: a 250 times molar excess of isothiocyanateDTTA-Eu (N1 chelate, WALLAC, Finland) was added to the Flt4 solution(0.5 mg/ml in PBS) and the pH was adjusted to about 9 by adding 0.5 Msodium carbonate buffer, pH 9.8. The labelling was performed overnightat +4° C. Unbound label was removed using PD-10 (PHARMACIA, Sweden) withTSA buffer (50 mM Tris-HCl, pH 7.8, containing 0.15 M NaCl) as eluent.

After purification, 1 mg/ml bovine serum albumin (BSA) was added to thelabelled Flt4 and the label was stored at +4° C. The average number ofEuropium ions incorporated per Flt4 molecule was 1.9, as determined bymeasuring the fluorescence in a ratio to that of known EuCl₃ standards(Hemmilä et al., Anal. Biochem., 137:335-343 (1984)).

The antibodies produced in Example 25 were screened using aSandwich-type immunofluorometric assay, using microtitration strip wells(NUNC, polysorb) coated with rabbit anti-mouse Ig (Z 259, DAKOPATTS).The pre-coated wells were washed once by Platewash 1296-024 (WALLAC)with DELFIA wash solution. The DELFIA assay buffer was used as adilution buffer for cell culture supernatants and for serum of thesplenectomized mouse (at dilutions between 1:1000 to 1:100,000) used aspositive control in the preliminary screening assay.

An overnight incubation at +4° C. (or alternatively for 2 hours at roomtemperature) was begun by shaking on a Plateshake shaker (1296-001,WALLAC) for 5 minutes followed by washing four times with wash solutionas described above.

The Europium-labelled Flt4 was added at a dilution of 1:500 in 100 μl ofthe assay buffer. After 5 minutes on a Plateshake shaker and one hourincubation at room temperature, the strips were washed as describedabove.

Enhancement solution (DELFIA) was added at 200 μl/well. The plates werethen shaken for 5 minutes on a Plateshake shaker and the intensity offluorescence was measured by ARCUS-1230 (WALLAC) for 10-15 minutes.(Lövgren et al., In: Collins W. P. (Ed.) Alternative Immunoassays, JohnWiley & Sons Ltd. (1985), pp. 203-216). The DELFIA results show that allmonoclonal antibodies tested bound the Flt4 EC antigen. Monoclonalantibodies reactive with the Flt4 (and the hybridomas which produce theantibodies) were selected for further screening.

The resulting monoclonal antibodies were used in double antibodyimmunofluorescence staining of NIH3T3 cells expressing the LTR-FLT41construct and neomycin-resistant transfected NIH3T3 cells. The cellswere detached from the culture plates using EDTA, stained, and analysedin a fluorescence-activated cell sorter (FACS). The results of FACSanalysis are given as percentages of cells staining positive with theindicated monoclonal antibody (see Table 2, below). TABLE 2 Mab clonesLTR %^(a)) NEO %^(b)) DELFIA-counts 1B1 67.3 1 20625 1B1D11 75 1.2 196941B1F8 76.1 1.4 18580 4F6 69.9 1.2 23229 4F6B8G12 75 0.3 24374 4F6B8H1175.9 0.3 28281 4F6B8E12 74.8 0.4 27097 4F6B8G10 75.3 0.4 26063 9D9 45.10.75 17316 9D9D10 71.7 2.3 18230 9D9F9 73 1.8 11904 9D9G6 74.3 2.9 167439D9G7 70.7 1.3 17009 10E4 24.2 1.4 39202 10E4B10E12 32.3 0.3 4249010E4B10G10 36.5 0.3 54815 10E4B10F12 45.6 0.4 43909 10E4B10G12 45.7 0.535576 11G2 30.2 1.6 11304 11G2D12 74.4 1.5 14660 11G2G9 74.2 0.9 1028311G2H7 74.4 2.1 25382^(a))FACS results with LTR transfected cells^(b))FACS results with NEO cells (control)

The FACS results with LTR-FLT41-transfected cells indicate that theantibodies effectively recognize Flt4-expressing cells. These sameantibodies give only background staining of neomycinphosphotransferase-trasfected NIH3T3 cells. Thus, the antibodiesspecifically recognize the Flt4 tyrosine kinase on the cell surface.

One clone, designated anti-Flt4 hybridoma 9D9F9, was found to stablysecrete monoclonal antibody which was determined to be of immunoglobulinclass IgG₁ by IFMA. Hybridoma 9D9F9 was deposited with the GermanCollection of Microorganisms and Cell Cultures, Department of Human andAnimal Cell Cultures and Viruses, Mascheroder Weg 1b, 3300 Braunschweig,Germany, Mar. 23, 1995, and given accession No. ACC2210.

Fusion II Antibodies

The Europium-labelled Flt4 extracellular domain protein described abovealso was used to screen the Fusion II antibodies described in Example25. The antibodies were screened using a Flt4-specific IFMA usingmicrotitration wells (Nunc, Polysorb) coated with rabbit anti-mouse Ig(Z 259, DAKO). The precoated wells were washed once with wash solution(Wallac) by using DELFIA Plate wash.

The DELFIA assay buffer was used as dilution buffer for cell culturesupernatants (dilution 1:2 in preliminary screening) and for serum ofthe splenectomized mouse (dilutions 1:1000 to 1:100,000) which was usedas a positive control. As standard, the purified anti-Flt4 9D9F9 (mousesubclass IgG₁) was used at concentrations between 1.0 ng/ml and 250ng/ml. Samples were first shaken at room temperature for five minutes ona Plateshake shaker and then incubated approximately 18 hours at +4° C.The frames were first washed four times, then the Eu-labelled Flt4(1:2000, in 100 μl assay buffer) was added, and finally the frames wereincubated for one hour at room temperature. After washing as described,the enhancement solution (200 μl/well, Wallac) was added, and the frameswere shaken for 5 minutes on the Plateshake shaker. The intensity offluorescence was measured by ARCUS-1230 (Wallac). Monoclonal antibodiesreactive with Flt4 were selected for further screening in the doubleantibody immunofluorescence staining assay employing Flt4-expressingNIH3T3 cells, as described above.

The resulting Fusion II monoclonal antibodies against Flt4 andcorresponding results of FACS analysis (expressed as percentages ofcells staining positive with the indicated monoclonal antibody) aresummarized in Table 3.

A standard curve for quantitation of anti-Flt4 antibodies was made byusing affinity purified anti-Flt4 9D9F9. The linear range reached from1.0 ng/ml to 250 ng/ml.

Cell lysate of NIH3T3 cells co-tranfected with pLTRFLT41 constructexpressing full-length Flt4 on the surface was electrophoresed in 6.5%SDS-PAGE, proteins were transferred onto nitrocellulose nitrate membrane(0.45 μm, SCHLEICHER & SCHUELL) and immunoblotted with monoclonalantibody-containing hybridoma cell culture supernatants (1:10, 50 mMTRIS-40 mM glycine buffer containing methanol 4%, SDS 0.04%). Thespecificities of monoclonal antibodies were detected using incubationwith HRP-conjugated rabbit antimouse Ig (P 161, DAKO, diluted 1:1000 in20 mM TRIS buffer, pH 7.5, containing 150 mM saline, 5% milk powder) andECL (Enhanced chemiluminescence, AMERSHAM). TABLE 3 approx. Mabproduction ng/ml/10⁶ Mab clones LTR %^(a)) NEO^(b)) cells^(c)) WB2B12E10 39.5 6.0 440 + 2E11D11 44.6 8.8 110 + 2E11F9 49.5 4.5 100 +2E11F12 46.0 4.1 180 + 2E11G8 41.2 7.8 160 + 6B2E12 NF NF 1390 + 6B2F8NF NF 470 + 6B2G6 NF NF 630 + 6B2H5 NF NF 740 + 6B2H8 NF NF 1800 +^(a))FACS results with LTR transfected cells^(b))FACS results with NEO cells (control)^(c))quantitation of Mab production based on affinity-purified antiFLT9D9F9 antibody used as standardNF not functioning in FACSWB Used successfully in Western immunoblotting

EXAMPLE 27 Use of Anti-Flt4 Antibodies to Identify Flt4 in Cell Lysatesand Expressed in Lymphatic Endothelial Cells in Human Tissue

The monoclonal antibodies produced by hybridoma 9D9 described in thepreceding examples were used in immunoprecipitation and Western blottingof lysates of HEL cells. As reported in Example 6, Flt4 mRNA expressionhad been previously observed in HEL cells. About 2×10⁷ cultured HELcells were lysed in RIPA buffer specified in Example 11 andimmunoprecipitated with about 2 micrograms of the 9D9 antibody (asdescribed for polyclonal antibodies in example 11). For Westernanalysis, immunoprecipitates were electrophoresed via SDS-PAGE (6% gel)and electroblotted onto a nitrocellulose membrane. Polypeptide bands of175 kD and 125 kD, corresponding to Flt4 polypeptides, were detected inthe Western blotting analysis of the immunoprecipitates using a 1microgram/ml dilution of the 9D9 antibody.

Immunostaining of human skin tissue was performed using the 9D9monoclonal antibodies and an alkaline phosphatase ABC-AP kit (Dako).Briefly, slides containing 6 μm samples of adult human skin were driedfor 30 minutes at room temperature (RT), fixed for ten minutes with coldacetone, and then washed once for five minutes with phosphate-bufferedsaline (PBS). The samples were then incubated for 30 minutes at RT with2% horse serum and washed three times for five minutes in PBS.

For immunostaining, the samples were incubated for one hour at RT withthe 9D9 primary antibody and washed three times for five minutes withPBS. After washing, the samples were incubated for thirty minutes at RTwith biotinylated rabbit anti-mouse secondary antibodies, and againwashed three times for five minutes with PBS.

Bound antibodies were detected by incubating the samples for thirtyminutes at RT with ABC-AP complex, washing three times with PBS,incubating for fifteen minutes at RT with AP-substrate (Sigma Fast RedTR/Naphtol AS-MX (Cat. No. F-4648)), and rinsing with water. Sampleswere then counter-stained with Mayer's hematoxylin for thirty secondsand rinsed with water. Aquamount and a coverslip were applied, and thesamples were analyzed under a mircoscope. The 9D9 antibody staining wasobserved in lymphatic endothelial cells in these human skin sections.Blood vessel endothelia showed extremely weak or no staining. Additionalanalyses have served to confirm the apparent specificity for lymphaticendothelia. See Lymboussaki et al., Am. J. Pathol., 153(2):395-403(August, 1998); and Jussila et al., Cancer Res., 58:1599-1604 (April,1998), both of which are incorporated herein by reference in theirentireties.

These results further confirm the utility of Flt4 as a useful marker forlymphatic endothelia and the utility of anti-Flt4 antibodies foridentifying and visualizing Flt4 expressed in these cells, in a tissuesample.

EXAMPLE 28 Upregulation of the VEGF-C/VEGFR-3 Signalling Pathway inBreast Cancer Angiogenesis

The foregoing examples demonstrate that Flt4 (VEGFR-3) is useful as aspecific antigenic marker for lymphatic endothelia in normal tissues.The following procedures additionally demonstrate that VEGFR-3 is usefulas an antigenic marker (e.g., for diagnosis and screening) and as atherapeutic target in malignant breast tumors. A highly elevated numberof VEGFR-3 positive vessels was found in invasive breast cancer incomparison to histologically normal breast tissue (P<0.0001).

Materials and Methods

Freshly frozen breast tissue samples were retrieved from the files ofthe Department of Pathology, University of Helsinki. The samplesconsisted of ductal carcinoma (n=6), lobular carcinoma (n=6),intraductal carcinoma (n=8), fibroadenoma (n=4), and histologicallynormal breast tissue (n=12). All samples had been frozen immediatelyafter surgical excision in liquid nitrogen, and stored at −70° C.

Mouse monoclonal antibodies (Mabs) against human Flt4 (VEGFR-3) wereproduced essentially as described in preceding examples, e.g., Example25. The VEGFR-3 extracellular protein domain (VEGFR-3EC) was expressedvia a recombinant baculovirus in insect cells, purified from the culturemedium. Mouse monoclonal antibodies against VEGFR-3EC were then producedusing standard methods and the immunoglobulin fraction was purified byprotein A affinity chromatography from hybridoma ascites fluid orTecnomouse® culture supernatants.

Five μm cryosections of the tissues samples were air-dried and fixed incold acetone for 10 minutes. The sections were re-hydrated in phosphatebuffered saline (PBS) and incubated for 30 minutes in 5% normal horseserum at room temperature. The sections were then incubated for 2 hoursin a humid atmosphere at room temperature with the Mabs 9D9F9 (Example26) at the concentration of 1.0 μg/ml. Other anti-VEGFR-3 Mab againstdistinct epitopes of the VEGFR-3EC were also studied; clones 2E11D11(Example 26) and 7B8F9 (made essentially as described in Example 26)were used at the concentrations of 9.5 and 8.5 μg/ml, respectively. Asubsequent incubation for 30 minutes in biotinylated anti-mouse serumwas followed by a 60 minute incubation using reagents of the VectastainElite Mouse IgG ABC kit (Vector laboratories, Burlingame, USA).Peroxidase activity was developed with 3-amino-9-ethyl carbazole (AEC,Sigma, St. Louis, USA) for 10 minutes. Finally, the sections werestained with haematoxylin for 20 seconds. Negative controls wereperformed by omitting the primary antibody, or by using irrelevantprimary antibodies of the same isotype. The purified baculoviralimmunogen was used to block the binding of the 9D9 antibodies as anothernegative control. In these experiments, the antibodies were incubatedovernight with a 10-fold molar excess of the VEGFR-3EC protein in PBS.After centrifugation for 4 minutes at 4000 rpm, +4° C., the supernatantwas carefully collected and then used as primary antibody. The 5 μmcryosections adjacent to the ones stained with the anti-VEGFR-3antibodies were immunostained for the blood vascular endothelial markerPAL-E (0.15 μg/ml, Monosan, Uden, the Netherlands), laminin (1:4000dilution of the supernatant of clone LAM-89, Sigma, St Louis, Mo.),collagen XVIII (1.9 μg/ml), α-smooth muscle actin (SMA, 0.5 μg/ml, clone1A4, Sigma), VEGFR-1 (1:200 dilution of the supernatant of clone 19) orVEGFR-2 (dilution 1:100).

Pathological examination of all of the samples was performed after thestaining procedures. The blood vascular densities were obtained from theslides stained for PAL-E [de Waal et al., Am. J. Pathol., 150: 1951-1957(1997)], following the guidelines recommended by Gasparini and Harris.[Gasparini G, and Harris A, J. Clin. Oncol., 13: 765-782 (1995).] TheVEGFR-3 positive vessel densities were studied in the same way. A slidewas first scanned at low magnification, and intratumoral vessel densitywas then assessed by counting the number of stained vessels per a 400×magnification high power field (hpf) in the areas with the highestvascular density (“vascular hotspots”) or in the areas with highestVEGFR-3 positive vessel density. A minimum of 5 fields was counted per aslide, after which the 3 highest counts were averaged.

Double staining was performed to differentiate immunohistochemicalstaining of lymphatic and blood vessels in two intraductal carcinomas.Acetone-fixed 5 μm cryosections were were incubated for 1 hour withanti-PAL-E antibodies, with biotinylated horse anti-mouse antibody(Vectastain Elite Mouse IgG ABC kit, Vector laboratories, Burlingame,USA) for 30 minutes, with ABC-peroxidase (Vectastain, 1:100) for 45minutes, and developed finally with AEC for 10 minutes. For the secondstep, the sections were incubated with anti-VEGFR-3 antibodies for 1hour (0.14 μg/ml), followed by biotinylated anti-mouse antibody for 30minutes (1:200 dilution of the supernatant of clone), ABC-peroxidase for30 minutes (1:100), biotinylated tyramin solution (1:2.000) containing0.01% peroxide for 5 minutes, ABC-alkaline phosphatase (1:100) for 20minutes, and developed with Fast Blue (Sigma, St. Louis, USA) for 20minutes, according to a procedure previously described in the literaturefor ISH signal enhancement. [Kerstens et al., J. Histochem. Cytochem.,43: 347-352 (1995).] Cryosections (5 μm) adjacent to the double-stainedsections were also immunostained with VEGFR-3 antibodies only, asdescribed above.

Polyclonal antibodies were produced in rabbits against a syntheticpeptide corresponding to the amino acid residues 2-18 of the N-terminusof mature, secreted human vascular endothelial growth factor C (VEGF-C)(residues 104-120 of the VEGF-C prepro-VEGF-C polypeptide) as describedin the literature. [Joukov et al., EMBO J, 16: 3898-3911 (1997),incorporated herein by reference in its entirety.] The antisera wereaffinity-purifed using the immunogenic polypeptide coupled to anepoxy-activated sepharose-6B column and tested for specific staining ofVEGF-C using cells infected with an adenoviral vector expressing VEGF-Cor control β-galactosidase.

The eight intraductal carcinomas and all of the invasive carcinomasanalysed for VEGFR-3 were chosen for further analyses of the expressionof VEGF-C. Five micrometer cryosections adjacent to the sections stainedwith the anti-VEGFR-3 antibodies were air-dried and fixed in coldacetone for 10 minutes. The sections were rehydrated in PBS andincubated for 30 minutes in 5% normal goat serum and then for 2 hours ina humid atmosphere at room temperature with the rabbit polyclonalantibodies against human VEGF-C, diluted 1:200 in PBS. A subsequentincubation for 30 minutes in biotinylated anti-rabbit serum was followedby a 60 minutes incubation using reagents of the Vectastain Elite RabbitIgG ABC kit (Vector laboratories, Burlingame, USA). The sections werefurther processed as described above. As a negative control, thepurified immunogen was used to block the binding of the VEGF-Cantibodies. In these experiments, VEGF-C antibodies were incubatedovernight with a 10-fold molar excess of the VEGF-C protein in PBS.After centrifugation for 4 minutes at 4000 rpm at +4° C., thesupernatant was carefully collected and used in the immunostainings.

Monoclonal antibodies to human type XVIII collagen were generated byDiaBor Ltd. (Oulu, Finland) by immunization of mice with the recombinantpolypeptide QH48.18 [Saarela et al., Matrix Biology, 16: 319-28 (1998)],corresponding to the common region of the N-terminal NC1 domain of humantype XVIII collagen. The clones were screened by ELISA assay and Westernanalysis using the polypeptide QH48.18, and also by immunofluorescencestaining of frozen human tissue sections. The screening of the hybridomaclones resulted in three monoclonal antibodies, which were positive inall three assays mentioned (ELISA, Western, immunofluorescencestaining). One of the antibodies which gave the strongest signals,DB144-N2, was used in subsequent experiments. It gave an identicalstaining pattern (e.g., in adult human skin and kidney samples) to thatof the polyclonal anti-all hu(XVIII).

Results

A. VEGFR-3 in Histologically Normal Breast Tissue and in BenignFibroadenomas

Immunohistochemical staining of VEGFR-3 in normal breast tissue showed avery weak staining in capillaries of the interductal stroma. Thesevessels did not form any specific pattern, but were scattered throughoutthe stroma. The density of the VEGFR-3 positive vessels in the normalbreast tissue samples ranged from 6 to 17 per hpf, median 9 (n=12). Mostof such vessels were strongly stained for the blood vascular endothelialmarker PAL-E and for the basal lamina component, collagen XVIII,suggesting that VEGFR-3 was expressed weakly in the blood vessels ofnormal breast tissue. However, some thin vessels in the stroma, whichwere clearly stained for VEGFR-3 were negative for PAL-E and only weaklypositive for the collagen type XVIII, suggesting that they werelymphatic vessels. VEGFR-3 positive vessels were also uniformly found inbenign fibroadenomas, where their density (median 8 vessels per hpf,range 3-19; n=4) did not differ from that of the histologically normalbreast tissue (median 8 vs. 9; P>0.1, the Mann-Whitney test).

B. VEGFR-3 Positive Vessels in Intraductal Carcinomas

In intraductal carcinomas, a distinctive pattern of strongly-stainedVEGFR-3 positive vessels was observed. The vessels formed arch-likestructures around the affected ducts (FIG. 5A). This “necklace” patternalso was observed in staining of adjacent sections for the blood vesselendothelial marker, PAL-E (FIG. 5B), suggesting that VEGFR-3 expressionwas enhanced in capillary endothelium. In order to more definitivelydifferentiate between blood and lymphatic vessels and to search for thepresence of smooth muscle cells and pericytes in the vessel walls,additional stainings were done using antibodies against smooth muscleα-actin (SMA) and basal lamina components laminin and type XVIIIcollagen. According to this staining, the small vessels close to theintraductal carcinomas expressed simultaneously VEGFR-3 and the basallamina proteins, but stained more weakly for SMA, indicating that theyare incompletely covered by pericytes/smooth muscle cells in the vesselwall (black arrows in FIGS. 5C-5F). In contrast, larger blood vessels atsome distance from the intraductal lesions were in general negative forVEGFR-3, but positive for laminin, collagen XVIII and SMA (red arrows).In addition, vessels were found, which were positive for VEGFR-3, butonly very weakly stained for the basal lamina proteins laminin and typeXVIII collagen and not at all for SMA (green arrows). These wereconsidered to represent lymphatic vessels.

C. Differential Double-Staining of Blood and Lymphatic Vessels

Two intraductal carcinomas were chosen for the immunohistochemicaldouble-staining procedure to more clearly differentiate lymphaticvessels from blood vessels. [See de Waal et al., Am. J. Pathol., 150:1951-1957 (1997).] Using this method, the VEGFR-3 positive vessels werestained blue, while the PAL-E positive vessels and basal laminae werestained brown. Both tested samples showed a similar pattern of staining:the vessels lining the tumor filled ducts were predominantly PAL-Epositive (arrowhead in FIGS. 5G and 5H) while the presumably lymphatic,VEGFR-3 positive vessels a short distance away in the interductal stromawere PAL-E negative (black arrows in FIGS. 5G and 5H). In order toexclude misinterpretation due to possible double-staining artefacts,adjacent 5 μm sections were stained with anti-VEGFR-3 alone. Thisstaining confirmed that several of the PAL-E positive blood vessles arealso positive for VEGFR-3.

D. VEGF-C, VEGFR-1, and VEGFR-2 in the Intraductal Carcinoma Cells andits Receptors in Adjacent Vessels

Polyclonal affinity-purified antibodies against human VEGF-C were usedto stain the 8 intraductal carcinoma samples. All tested samplescontained at least some VEGF-C, but considerable heterogeneity wasobserved in the intensity of staining and in the expression patterns. Insome cases, most of the carcinoma cells were strongly positive forVEGF-C, while in others, only some carcinoma cells gave a stainingsignal. In contrast, very little or no staining was observed in thenormal tissues surrounding the affected ducts, although weak signal wasalso obtained in unaffected normal ductal epithelium. Antigen blockingexperiments indicated that the staining for VEGF-C was specific. Theother VEGF-C receptor, VEGFR-2, as well as the other VEGF receptor(VEGFR-1), were both expressed in the same “necklace” vessels adjacentto the intraductal carcinoma cells.

E. VEGFR-3 Positive Vessels and VEGF-C in Invasive Breast Carcinoma

Strongly-stained VEGFR-3 positive vessels were also present in allinvasive ductal carcinomas and lobular carcinomas studied. The VEGFR-3positive vessels did not appear to form any specific distributionpattern; most of these vessels were also immunoreactive for the PAL-Eantigen. The intratumoral VEGFR-3 positive vessel density (median 21,range 9-56 vessels per hpf; n=12) was significantly elevated in theinvasive breast carcinomas when compared with normal breast tissue(median 21 vs. 9; P<0.0001, the Mann-Whitney test). Occasionally,invasion of the carcinoma cells into the VEGFR-3 positive lymphaticvessels could be observed.

Immunostaining for VEGF-C varied strongly among the invasive carcinomasstudied (n=12). Some carcinoma cells were strongly positive for VEGF-C,while others stained very weakly or, in some cases, no staining wasobserved. Like in the intraductal carcinomas, very little or no stainingwas observed in the connective tissue in these sections.

The foregoing data reveals that VEGFR-3, which had otherwise appeared tobe a predominantly lymphatic endothelial marker in most adult tissues,is very weakly expressed also in capillary endothelium of normal breasttissue. More significantly, in intraductal carcinomas, a “necklace”pattern of strongly-stained VEGFR-3 positive vessels was detected liningthe tumor-filled ducts. Most of these vessels expressed the blood vesselendothelial marker PAL-E and the basal lamina components laminin andcollagen XVIII, but apparently had less pericytes/smooth muscle cellsthan blood vessels located further away from the tumor cells, as shownby staining using antibodies against SMA. These features suggest thatthe “necklace” vessels were undergoing angiogenesis. A second group ofvessels lying a distance away from the affected ducts were positive forVEGFR-3 but very weakly positive for the basal lamina components andnegative for PAL-E, suggesting that they are lymphatic vessels. Thesevessels also lacked SMA-positive pericytic components. Also in invasivebreast carcinomas, VEGFR-3 was upregulated in PAL-E positive bloodvessels, although the vessel patterns seen were more randomly organizedin the connective tissue stroma around the tumor cells. The resultsindicate that VEGFR-3 expression is upregulated in breast carcinomasduring angiogenesis associated with tumor growth. The highly elevatednumber of VEGFR-3 positive vessels found in carcinoma in situ iscompatible with the hypothesis that the carcinoma cells produce factors,which activate the growth of blood vessels in the immediate vicinity ofthe carcinoma cells.

Since VEGF-C binds both VEGFR-3 and VEGFR-2 with high affinity, andsince both intraductal and invasive carcinoma cells often stainedpositive for VEGF-C protein, this growth factor is a candidate growthfactor for the VEGFR-3 and VEGFR-2 positive vessels in the carcinomas.These data are in agreement with another study, in which nearly half ofthirty-five unselected malignant invasive tumors (including breastcarcinomas, squamous cell carcinomas, lymphomas, melanomas, andsarcomas) contained VEGF-C mRNA in Northern blotting analyses. [SeeSalven et al., Am. J. Pathol., 153(1): 103-108 (July, 1998),incorporated herein by reference in its entirety.] Collectively, thedata reported herein provide an indication for treatment of breastcarcinomas and possibly other, non-lymphatic carcinomas with agents thatblock the VEGF-C mediated stimulation of VEGFR-3 and/or VEGFR-2.Contemplated blocking agents include: anti-VEGF-C antibodies;anti-VEGFR-3 antibodies; anti-VEGFR-2 antibodies; bispecific antibodiesthat bind to VEGFR-3 and either VEGFR-2 or VEGFR-1; solubleextracellular domain fragments of VEGFR-3 that will bind circulatingVEGF-C; VEGF-C fragments and analogs that bind VEGFR-3 and/or VEGFR-2and that inhibit activation of such receptors; VEGF-C polypeptides,fragments, and analogs that bind VEGFR-3 and/or VEGFR-2 and that areconjugated to a suitable therapeutic agent; VEGFR-3 tyrosine kinaseinhibitors; and small molecules that bind and inhibit these receptors.In addition, since VEGF-D binds both VEGFR-3 and VEGFR-2, it iscontemplated that anti-VEGF-D antibodies and inhibitory VEGF-D fragmentsand analogs are suitable blocking agents. Human or humanized antibodiesand fragments thereof are preferred, to the extent that antibody agentsare selected for human therapy. Additionally, it is contemplated, as anadditional aspect of the invention, to use any of the foregoing agentsto evaluate mammalian tissue in vitro or in vivo, e.g., for the purposesof diagnosis and screening for malignacies and the spread ofmalignancies.

For any of the foregoing agents, it is contemplated that the agent maybe further improved for diagnosis and screening by the attachment of adetectable label, including but not limited to radioisotopes (e.g., ¹⁴C,¹³³; and ¹²⁵I), chromophores (e.g., fluorescein, phycobiliprotien;tetraethyl rhodamine; enzymes which produce a fluorescent or coloredproduct for detection by fluorescence; absorbance, visible color, oragglutination, which produces an electron-dense product for detection byelectron microscopy); or electron dense molecules such as ferritin,peroxidase, or gold beads. Likewise, the agents may be further improvedfor therapeutic purposes by attachment (e.g., conjugation) orco-administration with molecules having anti-neoplastic properties, suchas toxins of plant, animal, microbial, or fungal origin; radioisotopes;drugs; enzymes; and/or cytokines and other therapeutic proteins. (See,e.g., Pietersz & McKenzie, “Antibody Conjugates for the treatment ofCancer,” Immunological Reviews, 129:57-80 (1992), incorporated byreference herein.

EXAMPLE 29 Anti-Flt4 Antibodies for Administration as a Therapeutic toHumans

A. Humanization of Anti-Flt4 Monoclonal Antibodies

The biology of Flt4 as reported herein, e.g., in Example 28, indicatestherapeutic uses for Flt4 inhibitors (antagonists) that blockligand-mediated signalling of the Flt4 receptor. Flt4-neutralizingantibodies comprise one class of therapeutics useful as Flt4antagonists. Following are protocols to improve the utility of anti-Flt4monoclonal antibodies as therapeutics in humans, by “humanizing” themonoclonal antibodies to improve their serum half-life and render themless immunogenic in human hosts (i.e., to prevent human antibodyresponse to non-human anti-Flt4 antibodies).

The principles of humanization have been described in the literature andare facilitated by the modular arrangement of antibody proteins. Tominimize the possibility of binding complement, a humanized antibody ofthe IgG4 isotype is preferred.

For example, a level of humanization is achieved by generating chimericantibodies comprising the variable domains of non-human antibodyproteins of interest, such as the anti-Flt4 monoclonal antibodiesdescribed herein, with the constant domains of human antibody molecules.(See, e.g., Morrison and Oi, Adv. Immunol., 44:65-92 (1989).) Thevariable domains of Flt4 neutralizing anti-Flt4 antibodies are clonedfrom the genomic DNA of a B-cell hybridoma or from cDNA generated frommRNA isolated from the hybridoma of interest. The V region genefragments are linked to exons encoding human antibody constant domains,and the resultant construct is expressed in suitable mammalian hostcells (e.g., myeloma or CHO cells).

To achieve an even greater level of humanization, only those portions ofthe variable region gene fragments that encode antigen-bindingcomplementarity determining regions (“CDR”) of the non-human monoclonalantibody genes are cloned into human antibody sequences. [See, e.g.,Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature,332:323-327 (1988); Verhoeyen et al., Science. 239:1534-36 (1988); andTempest et al., Bio/Technology, 9:266-71 (1991).] If necessary, theβ-sheet framework of the human antibody surrounding the CDR3 regionsalso is modified to more closely mirror the three dimensional structureof the antigen-binding domain of the original monoclonal antibody. (SeeKettleborough et al., Protein Engin., 4:773-783 (1991); and Foote etal., J. Mol. Biol., 224:487-499 (1992).)

In an alternative approach, the surface of a non-human monoclonalantibody of interest is humanized by altering selected surface residuesof the non-human antibody, e.g., by site-directed mutagenesis, whileretaining all of the interior and contacting residues of the non-humanantibody. See Padlan, Molecular Immunol., 28(4/5):489-98 (1991).

The foregoing approaches are employed using Flt4-neutralizing anti-Flt4monoclonal antibodies and the hybridomas that produce them, such asantibodies 9D9F9, to generate humanized Flt4-neutralizing antibodiesuseful as therapeutics to treat or palliate conditions wherein Flt4expression is detrimental.

B. Human Flt4-Neutralizing Antibodies from Phage Display

Human Flt4-neutralizing antibodies are generated by phage displaytechniques such as those described in Aujame et al., Human Antibodies,8(4):155-168 (1997); Hoogenboom, TIBTECH, 15:62-70 (1997); and Rader etal., Curr. Opin. Biotechnol., 8:503-508 (1997), all of which areincorporated by reference. For example, antibody variable regions in theform of Fab fragments or linked single chain Fv fragments are fused tothe amino terminus of filamentous phage minor coat protein pill.Expression of the fusion protein and incorporation thereof into themature phage coat results in phage particles that present an antibody ontheir surface and contain the genetic material encoding the antibody. Aphage library comprising such constructs is expressed in bacteria, andthe library is panned (screened) for Flt4-specific phage-antibodiesusing labelled or immobilized Flt4 as antigen-probe.

C. Human Flt4-Neutralizing Antibodies from Transgenic Mice

Human Flt4-neutralizing antibodies are generated in transgenic miceessentially as described in Bruggemann and Neuberger, Immunol. Today,17(8):391-97 (1996) and Bruggemann and Taussig, Curr. Opin. Biotechnol.,8:455-58 (1997). Transgenic mice carrying human V-gene segments ingermline configuration and that express these transgenes in theirlymphoid tissue are immunized with an Flt4 composition usingconventional immunization protocols. Hybridomas are generated using Bcells from the immunized mice using conventional protocols and screenedto identify hybridomas secreting anti-Flt4 human antibodies (e.g., asdescribed above).

D. Bispecific Antibodies

Bispecific antibodies that specifically bind to Flt4 and thatspecifically bind to other antigens relevant to pathology and/ortreatment are produced, isolated, and tested using standard proceduresthat have been described in the literature. See, e.g., Pluckthun & Pack,Immunotechology, 3:83-105 (1997); Carter et al., J. Hematotherapy, 4:463-470 (1995); Renner & Pfreundschuh, Immunological Reviews, 1995, No.145, pp. 179-209; Pfreundschuh U.S. Pat. No. 5,643,759; Segal et al., J.Hematotherapy, 4: 377-382 (1995); Segal et al., Immunobiology, 185:390-402 (1992); and Bolhuis et al., Cancer Immunol. Immunother., 34: 1-8(1991), all of which are incorporated herein by reference in theirentireties.

EXAMPLE 30 Animal Models to Demonstrate the Efficacy of Anti-Flt4Therapies for Treatment of Cancers

It is contemplated that any accepted animal for cancer therapies wouldbe useful to demonstrate the efficacy of anti-Flt4 therapies for cancertreatment. Exemplary models for demonstrating the efficacy for treatmentof breast cancers, using standard dose-response studies, include thosedescribed in Tekmal and Durgam, Cancer Lett., 118(1): 21-28 (1997);Moshakis et al., Br. J. Cancer, 43: 575-580 (1981); and Williams et al.,J. Nat. Cancer Inst., 66: 147-155 (1981). In addition to murine models,dog and pig models are contemplated because at least certain anti-humanFlt4 antibodies (e.g., the 9D9 antibodies) also recognize Flt4 from dogand pig. Tumor size and side effects are monitored to demonstratetherapeutic efficacy versus controls.

EXAMPLE 31 Soluble Flt4 Inhibits VEGF-C Mediated Tumor Growth andMetastasis

To further demonstrate the in vivo role of VEGF-C in tumorigenesis,MCF-7 human breast carcinoma cells overexpressing recombinant VEGF-Cwere orthotopically implanted into SCID mice. The VEGF-C overexpressionincreased tumor growth but, unlike VEGF-A overexpression, it had littleeffect on tumor angiogenesis. On the other hand, VEGF-C stronglypromoted the growth of tumor associated lymphatic vessels, which in thetumor periphery were commonly infiltrated with the tumor cells. Theseeffects of VEGF-C were inhibited by a soluble VEGFR-3 fusion protein.These data indicate that VEGF-C can upregulate tumor growth and/ormetastasis via the lymphatic vessels, and that these effects can beinhibited by blocking the interaction between VEGF-C and itsreceptor(s). In particular, a soluble VEGFR-3/Flt4 can be used to blockthis interaction.

Materials and Methods

A. Preparation of Plasmid Expression Vectors

The cDNAs coding for the human VEGF-C or VEGF₁₆₅ were introduced intothe pEBS7 plasmid (Peterson and Legerski, Gene, 107: 279-84, 1991.). Thesame vector was used for the expression of the soluble receptor chimerasVEGFR-3-Ig, containing the first three immunoglobulin homology domainsof VEGFR-3 fused to the Fc-domain of human immunoglobulin γ chain andVEGFR-1-Ig, containing the first five Ig homology domains of VEGFR-1 ina similar construct (Achen, et al., Proc Natl Acad Sci USA, 95: 548-53,1998).

B. Production and Analysis of Transfected Cells

The MCF-7S1 subclone of the human MCF-7 breast carcinoma cell line wastransfected with the plasmid DNA by electroporation and stable cellpools were selected and cultured as previously described (Egeblad andJaattela, Int J Cancer, 86: 617-25, 2000). The cells were metabolicallylabeled in methionine and cysteine free MEM (Gibco) supplemented with100 μCi/ml [³⁵S]-methionine and [³⁵S]-cysteine (Redivue Pro-Mix,Amersham Pharmacia Biotech). The labeled growth factors wereimmunoprecipitated from the conditioned medium using antibodies againstVEGF-C (Joukov, et al., EMBO J, 16: 3898-911, 1997) or VEGF (MAB293, R &D Systems). The immunocomplexes and the VEGFR-Ig fusion proteins wereprecipitated using protein A sepharose (Amersham Pharmacia Biotech),washed twice in 0.5% BSA, 0.02% Tween 20 in PBS and once in PBS andanalyzed in SDS-PAGE under reducing conditions.

C. Cell Proliferation and Tumorioenesis Assays

Cells (20 000/well) were plated in quadruplicate in 24-wells,trypsinized on replicate plates after 1, 4, 6, or 8 days and countedusing a hemocytometer. Fresh medium was provided after 4 and 6 days. Forthe tumorigenesis assay, sub-confluent cultures were harvested bytrypsination, washed twice and 10⁷ cells in PBS were inoculated into thefat pads of the second (axillar) mammary gland of ovariectomized SCIDmice, carrying subcutaneous 60-day slow-release pellets containing 0.72mg 17β-estradiol (Innovative Research of America). The ovarectomy andimplantation of the pellets were performed 4-8 days before tumor cellinoculation. Tumor length and width were measured twice weekly in ablinded manner, and the tumor volume was calculated as thelength×width×depth×0.5, assuming that the tumor is a hemi-ellipsoid andthe depth is the same as the width (Benz et al., Breast Cancer ResTreat, 24: 85-95, 1993).

D. Histology and Quantitation of the Blood Vessels

The tumors were excised, fixed in 4% paraformaldehyde (pH 7.0) for 24hours, and embedded in paraffin. Sections (7 μm) were immunostained withmonoclonal antibodies against PECAM-1 (Pharmingen), VEGFR-3 (Kubo etal., Blood, 96: 546-553, 2000) or PCNA (Zymed Laboratories) orpolyclonal antibodies against LYVE-1 (Banerji et al., J Cell Biol, 144:789-801, 1999), VEGF-C (Joukov et al., EMBO J, 16: 3898-911, 1997) orlaminin according to published protocols (Partanen et al., Cancer, 86:2406-12, 1999). The average of the number of the PECAM-1 positivevessels was determined from three areas (60× magnification) of thehighest vascular density (vascular hot spots) in a section. Allhistological analysis was performed using blinded tumor samples.

E. Adenoviral Expression of Soluble VEGFR-3 and Evan's Blue DrainingAssay

The cDNA coding for the VEGFR-3-1 g fusion protein was subcloned intothe pAdBglII plasmid and the adenoviruses produced as previouslydescribed (Laitinen et al., Hum Gene Ther., 9: 1481-6, 1998). TheVEGFR-3-Ig or LacZ control (Laitinen et al., Hum Gene Ther., 9: 1481-6,1998) adenoviruses, 10⁹ pfu/mouse, were injected intravenously into theSCID mice 3 hours before the tumor cell inoculation. After 3 weeks, fourmice from each group were narcotized, the ventral skin was opened and afew microliters 3% Evan's blue dye (Sigma) in PBS were injected into thetumor. The drainage of the dye from the tumor was followedmacroscopically.

Results

A. Expression of VEGF-C or VEGFR-3-Ig does not Affect MCF-7 Cell GrowthIn Vitro

The MCF-7 human breast carcinoma cells were transfected with expressionplasmids coding for full length human VEGF-C or a soluble VEGFR-3 fusionprotein (VEGFR-3-Ig) as described above and stable cell pools wereselected. For comparison, human VEGF₁₆₅ or VEGFR-1-Ig were expressed inthe same cells. Immunoprecipitation was used to analyze the conditionedmedia of these cells for the efficient production and secretion of theproteins. Immunoprecipitates of VEGF-C, VEGF or the soluble receptorproteins from metabolically labeled MCF-7 cells were analyzed in PAGEunder reducing conditions.

This investigation revealed that overexpression of VEGF-C, VEGF, solubleVEGFR-3 fusion protein or soluble VEGFR-1 fusion protein does not affectthe proliferation of the MCF-7 breast carcinoma cells in vitro. When thecells were seeded in 24-well plates and their growth was measured usinghemacytometer, it was found that the growth rate of the transfectedcells was not affected.

B. VEGF-C Increases Tumor Growth without Affecting Tumor Angiogenesis

To determine the in vivo effects of VEGF-C, the MCF-7 cell pools wereimplanted into the mammary fad pads of ovariectomized SCID mice carryingslow-release estrogen pellets to provide a constant level of the hormoneneeded to support the growth of the MCF-7 tumors.

Overexpression of VEGF-C increased tumor growth significantly (VEGF-C:545 mm³±110 mm³, control: 268 mm³±69 mm³ at 13 days, n=8, p<0.0001,Student's t-test). However, the effect of VEGF-C overexpression on tumorgrowth was much less dramatic than that of VEGF-A (VEGF-A: 1136 mm³±339mm³, control: 189 mm³±57 mm³, at 15 days, n=6, p<0.0001, Student'st-test). The increased tumor growth was neutralized by mixing the VEGF-Cor VEGF overexpressing MCF-7 cells with cells expressing the solubleVEGFR-3 or VEGFR-1 fusion proteins, respectively. Further, it was foundthat the increased growth of the VEGF-C overexpressing tumors also wasinhibited by a circulating soluble VEGFR-3-1 g expressed in the liver byan intravenously injected recombinant adenovirus.

To investigate the effect of VEGF-C on tumor angiogenesis, sections ofthe tumors were stained for PECAM-1, an endothelial antigen primarilyexpressed in blood vessels and only weakly in lymphatic vessels.Quantitation of the PECAM-1 positive vessels in the tumors revealed thatoverexpression of VEGF-C had very little effect on the density of thetumor blood vessels (40.2±12.2 vessels/microscopic field for VEGF-Ctumors, n=18 and 36.6±11.6 for control tumors, n=23, average of threedifferent experiments). In contrast, overexpression of VEGF-A increasedthe vascular density approximately two-fold.

C. VEGF-C Overexpression is Associated with Lymphangiogenesis andIntralymphatic Growth of Tumor Cells

The effect of VEGF-C on tumor associated lymphatic vessels was analysedby immunostaining for the lymphatic specific marker LYVE-1 (Banerji etal., J Cell Biol, 144: 789-801, 1999.). This marker revealed highlyhyperplastic lymphatic vessels in the periphery of the VEGF-Coverexpressing tumors. The proliferating cell nuclear antigen (PCNA) wasdetected in many of the LYVE-1 positive endothelial cells, showing thatthese lymphatic vessels were actively proliferating. Confirmation of thelymphatic identity of the vessels was obtained by staining for VEGFR-3and by the lack of staining for the basal lamina component laminin. Thinlymphatic vessels were also present inside some of the VEGF-Coverexpressing tumors.

The lymphatic vessels in the tumor periphery were commonly infiltratedby the VEGF-C positive tumor cells. In a striking contrast, the VEGFoverexpressing and control tumors contained no or only few lymphaticvessels.

D. VEGF-C Induced Lymphangiogenesis is Inhibited by a CirculatingSoluble VEGFR-3 Fusion Protein

In human breast cancer, the centinel node method is used to tracelymphatic drainage and metastatic spread (for a review, see Parker etal., Radiol Clin North Am, 38: 809-23, 2000). In order to tracelymphatic drainage of the MCF-7 tumors, Evan's blue dye was injectedinto VEGF-C overexpressing or control tumors in mice infected withVEGFR-3-Ig or control adenovirus. Control experiments indicated thatinfection of cultured human embryonic kidney cells with the VEGFR-3-Igadenovirus resulted in the secretion of high amounts of the solubleVEGFR-3-Ig fusion protein and intravenous infection of mice led to highsystemic levels of the VEGFR-3-Ig fusion protein in the serum. Injectionof Evan's blue dye into the tumors resulted in the staining of lymphaticbut not blood vessels and revealed an increased number of enlargedlymphatic vessels surrounding the VEGF-C overexpressing tumors whencompared to control tumors. Most of the enlarged lymphatic vessels wereabsent from VEGF-C overexpressing tumors in mice treated with theVEGFR-3-Ig adenovirus.

The foregoing data demonstrate that VEGF-C overexpression in MCF-7mammary tumors strongly and specifically induces the growth of tumorassociated lymphatic vessels, but does not have major effects on tumorangiogenesis. Furthermore, it demonstrated that VEGF-C-mediatedincreased tumor growth and tumor-associated lymphangiogenesis wereinhibited by a soluble VEGFR-3 fusion protein, i.e., an agent selectedto block VEGF-C-mediated stimulation of endothelial cells that expressVEGFR-3.

Due to the lack of specific markers, it has in the past been difficultto determine whether tumors can actively induce lymphangiogenesis ormerely encompass the already existing lymphatic vessels by overgrowthand compress them due to the high interstitial fluid pressure inside thetumor. Data from various experimantal models has recently suggested thelatter occurs (Leu et al., Cancer Res, 60: 4324-7, 2000; Stohrer et al.,Cancer Res, 60: 4251-5, 2000.). Here, for the first time, it is shownthat overexpression of VEGF-C can induce the growth of lymphatic vesselsin association with experimental tumors. The VEGF-C induced lymphaticvessels in the tumor periphery were highly hyperplastic and mostlyfilled with tumor cells, whereas the lymphatic vessels inside the tumorwere flattened and without a lumen. Unlike lymphatic endothelial cellsin normal adult tissues, the lymphatic endothelial cells associated withthe MCF-7 tumors were actively proliferating. Thus, it would appear thatmost of the peri- and intratumoral lymphatic vessels were generated byproliferation of the endothelial cells of pre-existing lymphaticvessels.

The spread of cancer through the lymphatics into the regional lymphnodes has long been an important prognostic indicator in clinical use.The growth of tumor cells inside the enlarged lymphatic vesselsassociated with the VEGF-C-overexpressing tumors as demonstrated in thisExample, closely resembles the peritumoral lymphatic invasion, thatcorrelates with metastatic spread to the lymph nodes and poor survivalin human breast cancer (Lauria et al., Cancer, 76: 1772-8, 1995). Thus,the data reported herein provides evidence that expression of VEGF-Cpromotes tumor metastasis via the lymphatic system. Thus, whereasExample 28 and other data indicates that VEGFR-3 is upregulated in theblood vessels of many kinds of solid tumors (Valtola et al., Am JPathol, 154: 1381-90, 1999; Partanen et al., Cancer, 86: 2406-12, 1999;Kubo et al., Blood, 96: 546-553, 2000), the present Example demonstratesa tumor model wherein overexpression of VEGF-C effects were mainlylymphangiogenic.

The effect of VEGF-C on tumor growth was not simply due to variationbetween the cell pools, as shown by the ability of the VEGFR-3 fusionprotein to inhibit the growth of VEGF-C overexpressing tumors. Byinjecting Evan's blue dye into the tumors, it was demonstrated that anincreased number of large draining lymphatic vessels were associatedwith the VEGF-C overexpressing tumors. It is possible that the highernumber of functional lymphatic vessels may result in a better lymphaticdrainage and thus a lower interstitial pressure and enhanced bloodperfusion of the VEGF-C overexpressing tumors. Irrespective of whetherVEGF-C/D-VEGFR-3 mediated tumor progression for a particular tumorproceeds through angiogenesis lymphangiogenesis, or both, thetherapeutic methods of the invention should inhibit these processes.

In conclusion, the results above show that VEGF-C produced by tumorcells can induce the growth of lymphatic vessels around tumors and thusfacilitate the intralymphatic spread of cancer. The data indicates thatinhibition of tumor associated lymphangiogenesis, for example by genetherapy employing soluble VEGFR-3 proteins, represents a valuable way ofinhibiting tumor metastasis.

EXAMPLE 32 Inhibition of Lymphangiogenesis in Mice Expressing SolubleVEGFR-3/Flt4

The previous Example demonstrated that VEGF-C increased in vivo tumorgrowth and this promotion of tumor growth of tumor was associatedlymphatic vessels. These effects of VEGF-C were inhibited by a solubleVEGFR-3 fusion protein. The present Example provides further evidencethat soluble VEGFR-3 is a potent inhibitor of VEGF-C/VEGF-D signalingand, when expressed in the skin of transgenic mice, it inhibitslymphangiogenesis and induces a regression of already formed lymphaticvessels while the blood vasculature remains intact.

More particularly, the present example shows that a chimeric proteinconsisting of the ligand-binding portion of the extracellular portion ofVEGFR-3, joined to the Fc domain of immunoglobulin (Ig) γ-chain(VEGFR-3-Ig) neutralizes the activity of VEGF-C and VEGF-D and inhibitsthe formation of the dermal lymphatic vasculature when expressed inmouse epidermis under the keratin-14 (K14) promoter. As the blood vesselnetwork remained normal in these mice, the inhibition appears to bespecific to the lymphatic vessels. VEGFR-3-Ig induced regression oflymphatic vessels during embryonic development, indicating thatcontinuous signaling by this receptor is essential for the maintenanceof the lymphatic vasculature.

Materials and Methods

A. Production of VEGF-C, VEGF-D and VEGFR-Ig Fusion Proteins

The mature form of human VEGF-C as described above in Example 31 and byJoukov et al., (EMBO J. 16:3898-3911, 1997). VEGF-D was obtained fromR&D Systems (Minneapolis, Minn.). The VEGFR-1-Ig and VEGFR-3-Ig proteinsconsisting of the ligand-binding domains of human VEGFRs fused to humanIgG1 Fc domain were produced in Drosophila S2 cells (Invitrogen,Carlsbad, Calif.).

B. VEGFR-3 Bioassay

Ba/F3 cells expressing the VEGFR-3/EpoR chimera (Achen, Eur. J. Biochem.267, 2505-2515, 2000) were seeded, in 96-well microtiter plates at15,000 cells/well in triplicates supplied with 100 ng/ml of VEGF-C andwith indicated concentrations of VEGFR-Ig proteins. After 48 h, theviability of the cells was determined by adding MTT(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (Sigma),0.5 mg/ml), followed by further 2 h of culture, addition of an equalvolume of cell lysis solution (10% SDS, 10 mM HCl) and incubationovernight at 37° C. Absorbance was measured at 540 nm.

C. Generation of the Transgenic Mice

The sequence encoding human VEGFR-3 Ig-homology domains 1-3 wasamplified using PCR. The primers employed for this purpose were:5′-TACAAAGCTTTTCGCCACCATGCAG-3′ (SEQ ID NO:23) and5-TACAGGATCCTCATGCACAATGACCTC-3′ (SEQ ID NO:24).

The PCR product was cloned into the plg-plus vector (Ingenius, R&DSystems) in frame with human IgG1 Fc tail. The VEGFR-3-Ig construct wasthen transferred into the human keratin-14 promoter-expression vector.The expression cassette fragment was injected into fertilized mouseoocytes of the FVB/NIH and DBAxBalbC hybrid strains to create sevenlines of K14-VEGFR-3-Ig mice. Transgene expression was analyzed and thephenotype was confirmed from all three founder lines expressing thetransgene as described below.

D. Analysis of Transgene Expression

For northern blotting, 10 μg of total RNA extracted from skin in 1%agarose was subjected to electrophoresis, transferred to nylon filters(Nytran), hybridized with the corresponding [³²P]-labeled cDNA probesand exposed autoradiography. For western blotting, skin biopsies werehomogenized into the lysis buffer (20 mM Tris, pH 7.6, 1 mM EDTA, 50 mMNaCl, 50 mM NaF, 1% Triton-X100) supplemented with 1 mM PMSF, 1 mU/mlapprotinin, 1 mM Na₃VO₄ and 10 μg/ml leupeptin. The Ig-fusion proteinswere precipitated from 1 mg of total protein and separated in SDS-PAGE,transferred to nitrocellulose and detected using the horseradishperoxidase conjugated rabbit antibodies against human IgG (DAKO,Carpinteria, Calif.) and the enhanced chemiluminescence detectionsystem.

E. Immunohistochemistry and TUNEL Staining

Paraffin sections (5 μm) from 4% paraformaldehyde (PFA) fixed tissueswere stained using rat monoclonal antibodies against mouse VEGFR-3 (Kuboet al., Blood 96:546-553, 2000) or CD31I/PECAM-1 (PharMingen, San Diego,Calif.), rabbit polyclonal antibodies against mouse LYVE-1 (Banerji etal., J Cell Biol, 144: 789-801, 1999), or biotinylated mouse monoclonalantibodies against human IgG Fc domain (Zymed, San Diego, Calif.). ForTUNEL staining, detection of DNA fragmentation was done using in situCell Death Detection Kit (fluorescein; Roche, Indianapolis, Ind.).

F. Visualization of Blood and Lymphatic Vessels

For visualization of blood vessels (Thurston et al. Science 286,2511-2514 (1999), 100 μl of 1 mg/ml biotinylated Lycopersicon esculentumlectin (Sigma) was injected (IV) by the femoral vein and allowed tocirculate for 2 min. After fixation by perfusion with 1% PFA/0.5%glutaraldehyde in PBS, bound lectin was visualized by theABC-3,3″-diaminobenzidine peroxidase reaction. In VEGFR-3+/LacZ mice thelymphatic vessels were then stained by the β-galactosidase substrateX-Gal (Sigma, St. Louis, Mo.). For the visualization of functionallymphatic vessels, Evans blue dye (5, mg/ml; Sigma, St. Louis, Mo.) wasinjected into the footpad of the hindlimb or TRITC-dextran (Sigma, 8mg/ml) was injected into the ear or tail and the lymphatic vessels wereanalysed by light or fluorescence microscopy, respectively.

G. Detection of the VEGFR-3-Ig Protein in Serum

ELISA plates (Nunc Maxisoip, Copenhagen, Denmark) were coated with mouseantibodies against human IgG (Zyrned, 2 μg/ml in PBS) or human VEGFR-3(clone 7B8, 4 μg/ml). The mouse sera were diluted into the incubationbuffer (5 mg/ml BSA, 0.05% Tween 20 in PBS) and allowed to bind for 2 hat room temperature. The plates were then washed 3 times with incubationbuffer before addition of mouse anti-human IgG1 (Zymed, 1:500) for 1 h.Streptavidin conjugated with alkaline phosphatase (Zymed, 1:5000) wasthen incubated in the wells for 30 min, followed by addition of thesubstrate (1 mg/ml p-Nitrophenyl phosphate in 0.1 M diethanolamine, pH10.3) and absorbance reading at 405 nm.

H. Magnetic Resonance Imaging

MRI data was acquired using a s.m.i.s. console (Surrey Medical ImagingSystems, Guildford, UK) interfaced to a 9.4 T vertical magnet (OxfordInstruments, Oxford, UK). A single loop surface coil (diameter 35 mm)was used for signal transmission and detection. A T₂-weighted (TR 2000ms, TE 40 ms, 4 scans/line) multislice spin-echo sequence was used withan FOV of 25.6 mm² (matrix size: 256×128) and slice thickness of 1.3 mmin transverse orientation. Saturation pulses centered at 1.2 ppm wereused to decrease fat signals in T₂-images. Diffusion weighted MRI wasacquired using monopolar diffusion gradients (b-value=800 s/mm²) alongslice axis in the spin-echo sequence (TR 2000 ms, TE 40 ms), and waterapparent diffusion coefficient (ADC) was computed by fitting the MRIdata as function of b-values into a single exponential.

Results

A. Soluble VEGFR-3 Inhibits VEGF-C-Mediated Signaling In Vitro

To inhibit VEGF-C signaling through VEGFR-3, a fusion protein consistingof the first three Ig-homology domains of VEGFR-3 and IgG Fc domain wasemployed. The VEGFR-3-Ig bound VEGF-C and VEGF-D with the sameefficiency as the full-length extracellular domain and inhibitedVEGF-C-induced VEGFR-3 phosphorylation and subsequent p42/p44mitogen-activated protein kinase (MAPK) activation in VEGFR-3 expressingendothelial cells. In contrast, a similar VEGFR-1-Ig fusion protein,which does not bind VEGF-C, did not affect p42/p44 MAPK activation.

The effect of soluble VEGFR-3 on VEGF-C signaling also was determined ina bioassay using a chimeric VEGFR-3/erythropoietin (Epo) receptorcapable of transmitting VEGF-C dependent survival and proliferationsignals for the IL-3 dependent Ba/F3 cells in the absence of IL-3 (Achenet al., Eur. J. Biochem., 267:2505-2515, 2000). In this cellular assay,there was a complete inhibition of VEGF-C-dependent cell survival at a0.5:1 molar ratio (VEGFR-3-Ig:VEGF-C), whereas VEGFR-1-Ig had no effect.Similarly, VEGFR-3-Ig also abolished VEGF-D-induced survival of theVEGFR-3/EpoR cells. In contrast, even a ten-fold molar excess ofVEGFR-2-Ig only partially abolished VEGF-C dependent viability, perhapsbecause of lower affinity of VEGF-C to VEGFR-2.

B. Soluble VEGFR-3 Inhibits the Formation of Lymphatic Vessels In Vivo

To determine the inhibitory effect of VEGFR-3-Ig in vivo, the fusionprotein was expressed under the control of K14 promoter, which directstransgene expression to the basal epidermal cells of the skin.VEGFR-3-Ig expression was detected in mice by northern blotting of skinRNA and by western blotting of protein extracts from the skin. Thesemice appeared healthy and fertile and had a normal lifespan.Histological examination of the skin revealed a thickened dermis andsubcutaneous layer. Antibody staining confirmed VEGFR-3-Ig expression inthe basal keratinocytes. When the skin sections were stained for markersof the lymphatic endothelium, VEGFR-3 (Jussila et al., Cancer Res.58:1599-1604, 1998; Kubo et al., Blood, 96 546-553, 2000) and LYVE-1(Banerji et al., J Cell Biol, 144: 7S9-801, 1999), no lymphatic vesselswere observed in the transgenic mice, even though lymphatic vessels werestained in the skin of control mice. In contrast, blood vessels werestained for the panendothelial marker PECAM-1/CD31 in both transgenicand wild-type skin.

C. Soluble VEGFR-3 Suppresses Lymphangiogenesis but not Angiogenesis

In order to visualize better the lymphatic vessels, the K14-VEGFR-3-Igmice were mated with heterozygous VEGFR-3+/LacZ mice that express β-galin the Flt4 locus (Dumont et al. Science 282, 946-949, 1998). Whenwhole-mount tissue preparations of the ear skin were stained using thesubstrate X-gal, no lymphatic vessels were detected, whereas, in thecontrol mice, blue-staining lymphatic vessels were visualized. Invascular perfusion staining using biotin-labeled lectin (Thurston et al.Science 286, 2511-2514, 1999), the blood vessels appeared normal in theK14-VEGFR-3-Ig mice.

The absence of the lymphatic vessels also was confirmed using afunctional assay, monitoring the fate of Evans blue dye or TRITC-dextraninjected into the skin. The dye was rapidly collected into the lymphaticvessels surrounding the ischiatic vein after injection into the hindlimbfootpads of wild-type mice, whereas no dye was seen in such vessels inthe transgenic mice where collecting lymphatic vessels were eitherabsent or rudimentary. The lymphatic vessels in control mice also werevisualized using fluorescence microscopy for TRITC-dextran injectedintradermally into the ear or tail, whereas no such vessels were seen inthe transgenic mice.

D. Circulating Soluble VEGFR-3 is Associated with a Transient Loss ofLymphatic Tissue in Internal Organs

By the age of two weeks, the VEGFR-3-Ig/VEGFR-3+/LacZ mice had only afew thin and rudimentary, if any, lymphatic vessels in organs such asdiaphragm, heart, lungs, caecum, pancreas, mesenterium and esophaguswhen compared with the control VEGFR-3+/LacZ littermate mice. Suchfindings, obtained by X-Gal staining, were confirmed by immunostainingfor VEGFR-3 and LYVE-1. In addition, the lack of lymphatic vessels inheart pericardium was associated with pericardial fluid accumulation inat least some of the mice. At three weeks of age, regrowth of thelymphatic vessels was apparent. In adult transgenic mice, only someorgans such as heart and diaphragm had abnormally patterned andincompletely developed lymphatic vessels.

The effects seen in the internal organs indicate that the solubleVEGFR-3-Ig protein circulates in the bloodstream. Indeed the fusionprotein was detected in the serum of the transgenic mice using aspecific enzyme-linked immunosorbent assay; the levels ranged between100-200 ng/ml, being highest in the young mice. Based on our in vitroexperiments, such concentrations would neutralize about 20-40 ng/ml ofVEGF-C. The VEGFR-3-Ig protein was relatively stable in the bloodstream,as intravenously injected recombinant VEGFR-3-Ig was in the serum for atleast nine hours.

E. The Transgenic Phenotype has Features of Human Lymphedema

The K14-VEGFR-3-Ig mice were distinguished from their wild-typelittermates by the swelling of their feet, which was already visible atbirth. Older mice showed thickening of the skin, dermal fibrosis andincreased deposition of subcutaneous fat. Magnetic resonance imaging(MRI) revealed prominent T₂-hyperintense regions in foot skin andsubcutaneous tissues of the transgenic mice indicating increased fluidaccumulation, whereas similar regions were absent in littermatecontrols. The apparent diffusion coefficient (ADC) for thesehyperintense areas was 1.99 [0.60]×10⁻³ mm²/s, being higher than fornormal tissue where ADC was 1.32 [0.21]×10⁻³ mm²/s, and about 1-2 ordersof magnitude greater than the values for fat (Thurston, et al. Science286, 2511-2514 (1999). In addition, size and appearance of lymph nodesvaried, especially in the large para-aortic lymph nodes surrounding theinferior vena cava. However, mesenteric lymph nodes and Peyer patcheswere seen in the VEGFR-3-Ig mice.

F. Regression of the Developing Lymphatic Vessels by Endothelial CellApoptosis

During embryogenesis, a dramatic increase in K14-driven transgeneexpression occurs at E14.5, and by E16.5 the expression encompasses thewhole embryonic skin (Byrne, et al., Development 120, 2369-2383, 1994).When analyzed in the VEGFR-3+/LacZ background by X-Gal staining, thelymphatic networks of the skin were indistinguishable between transgenicand wild-type embryos at E15. At E15.5-16.5, the lymphatic vessels ofthe transgenic embryos had regressed in some areas. At E17.5, thelymphatic vessels still formed a continuous network but were thinnerthan in control embryos. At E18.5, the whole cutaneous lymphatic networkwas disrupted in the transgenic embryos and after birth, none or only afew single disrupted lymphatic vessels were in the skin, mainlyaccompanying the large dermal blood vessels. Thus, the lymphatic vesselsinitially form in the skin during embryogenesis, but regress when theexpression of the transgene is turned on. However, the formation of thedermal blood vasculature was not inhibited in the K14-VEGFR-3-Ig embryosas shown by X-Gal staining in the Tie-promoter-LacZ background (Korhonenet al. Blood 86, 1828-1835 (1995).

TUNEL staining was used to detect apoptosis in endothelial cells, whichwere identified by simultaneous staining for PECAM-1. Apoptoticendothelial cells were seen in the dermis of the transgenic embryosfirst at E17.5 and E18.5. No endothelial cell apoptosis was seen inwild-type embryos. The TUNEL-positive cells were detected almostexclusively in VEGFR-3 positive endothelia in the transgenic skin,indicating that VEGFR-3-1 g mediated apoptosis was targeted to thelymphatic endothelium.

The present Example shows that soluble VEGFR-3 fusion protein inhibitslymphangiogenesis and leads to regression of existing fetal lymphaticvessels in vivo. Continuous VEGFR-3 signaling is thus essential for thefetal development and maintenance of the lymphatic vascular system.

The absence of lymphatic vessels in the skin of K14-VEGFR-3-Ig mice wasassociated with a thickening of the dermis and especially thesubcutaneous fat layer as in human lymphedema—a disorder caused byinsufficiency of the lymphatic system and characterized by swelling ofthe extremities of increasing severity (Witte et al., Lymphangiogenesis:Mechanisms, significance and clinical implications, in Regulation ofAngiogenesis (eds. Goldberg, I. D. & Rosen, E. M.) 65-112 (BirkhäuserVerlag, Basel, Switzerland) 1997; Mortimer, Cancer 83, 2798-2802 (1998).In primary lymphedema, which is an inherited disease, the superficial orsubcutaneous lymphatic vessels are usually hypoplastic or aplastic, andthey fail to transport the lymphatic fluid into the venous circulation.Noninherited secondary or acquired lymphedema develops when thelymphatic vessels are damaged by surgery, radiation, infection ortrauma. In lymphedema, a protein-rich fluid accumulates in interstitialspace, leading to tissue fibrosis and adipose degeneration, interferencewith wound healing, and susceptibility to infections. In K14-VEGFR-3-Igmice, there was a lack of macromolecular transport in the dermis and,especially in older mice, signs of dermal fibrosis. Moreover, theswelling of the feet and increased fluid accumulation in the skin andsubcutaneous tissue in the transgenic mice were similar to symptoms ofhuman lymphedema. The skin phenotype of the K14-VEGFR-3-Ig mice thusshares several features with human lymphedema. In studies of somelymphedema families, heterozygous inactivating missense mutations havebeen detected in the tyrosine kinase encoding region of Flt4 (Karkkainenet al., Nature Genet., 25:153-159, 2000; Irrthum et al., Am. J. Hum.Gen. 67:259-301 2001). At least some lymphedema patients havedysfunctional lymphatics due to defective VEGFR-3 signaling. Inconcurrence with this observation, the results in this Example show thatthe disruption of VEGFR-3 signaling by the soluble VEGFR-3 protein cancompletely destroy the lymphatic network and lead to a lymphedema-likephenotype. Moreover, as in some cases of lymphedema, the size andappearance of certain regional lymph nodes was variable, indicating thatlymph flow and a functional lymphatic vasculature are essential for theformation of normal lymph nodes.

VEGFR-3-Ig also induced regression of the already-formed lymphatics.Thus, inhibition of VEGF-C and/or VEGF-D binding to VEGFR-3 duringdevelopment leads to apoptosis of the lymphatic endothelial cells and tothe disruption of the lymphatic network, which indicates that continuousVEGFR-3 signaling is required for the survival of the lymphaticendothelial cells. In cell culture, VEGFR-3 activates biochemicalsignaling cascades associated with endothelial cell survival. Althoughtransgenic mice that overexpress either VEGF-C (Jeltsch et al., Science,276:1423-1425, 1997) or VEGF-D in the skin develop a hyperplastic dermallymphatic vasculature, their dermal lymphatic vessels also regress whenmated with K14-VEGFR-3-Ig mice. As both VEGFR-3 ligands are alsoexpressed in the skin, the phenotype observed in K14-VEGFR-3-Ig micemight be due to a simultaneous inhibition of both VEGF-C and VEGF-D.

Although VEGF-C and VEGF-D are mitogenic for blood vascular endothelialcells both in vitro and in vivo (Joukov et al. EMBO J. 16, 3898-3911,1997; Achen et al., Proc. Natl. Acad. Sci. USA 95, 548-553, 1998; Cao etal., Proc. Natl. Acad. Sci. USA 95, 14389-14392 1998; Witzenbichler etal., Am. J. Pathol 153, 381-394, 1998; Marconcini et al., Proc. Natl.Acad. Sci. USA 96, 9671-9676, 1999), VEGFR-3-Ig did not seem to affectthe blood vessels. The late onset of K14-promoter expression may explainthe lymphatic specificity of the VEGFR-3-Ig protein. A substantialincrease in K14-promoter activity is not seen until around E14.5-16.5(Byrne et al., Development 120:2369-2383, 1994). Although the expressionof endogenous VEGFR-3 is first detected at E8.5 in developing bloodvessels, by E14.5-16.5 it has been largely down-regulated in healthyblood vascular endothelia (Dumont et al., Science 282, 946-949 1998;Kaipainen, et al., Proc. Natl. Acad. Sci. USA 92, 3566-3570, 1995).Therefore, during the developmental period when VEGFR-3 no longer occursnormally in the blood vessel endothelium of healthy tissues, VEGFR-3signaling plays a more minimal role in angiogenesis in the skin than doother receptors.

In young VEGFR-3-Ig mice, several internal organs were almost completelydevoid of lymphatic vessels, but they regrew in adult mice, althoughinto an abnormal pattern in some organs. The growth and maintenance oflymphatic vasculature can therefore be reactivated in adult organs.Decreasing levels of VEGFR-3 inhibition or independent signals from, forexample, maturing connective tissue matrix might have reactivatedlymphangiogenesis, but there was no evidence obtained that would suggestthat increased VEGF-C or VEGF-D levels were responsible. However,administration of VEGF-C through an adenovirus vector in quantitiesexceeding those usually found in interstitial fluids can lead tolymphatic growth in adult tissues. The use in gene therapy of a modifiedVEGF-C that no longer binds VEGFR-2 (U.S. Pat. No. 6,130,071 Joukev etal., J. Biol. Chem., 273:6599-6602) might prevent its potential effectson the blood vascular endothelium.

Thus, soluble VEGFR-3 is a potent and specific inhibitor oflymphangiogenesis in vivo. The soluble VEGFR-3 may comprise theextracellular fragment of Flt4 as described elsewhere in thespecification. As seen above a preferred soluble VEGFR-3 is one whichcomprises the first three domains of VEGFR-3, however it should beunderstood that the soluble VEGFR-3 may be a fragment of VEGFR-3 whichcomprises more or less of the wild-type sequence of VEGFR-3 that isdepicted in FIG. 2. For example, the soluble peptide also may compriseone or more of IgIV, IgV, IgVI, IgVII. Alternatively, it may be that asoluble VEGFR-3 may comprise only IgI in any combination with one ormore of the domains selected from the group consisting of IgII, IgIII,IgIV, IgV, IgVI and IgVII.

In addition, the present Example also establishes a mouse model that hasfeatures of human lymphedema. As lymphedema always involves the skin,this mouse model is useful in understanding and characterizing thisdisease and in testing of new therapies that could be applied to humanpatients.

All documents including patents and journal articles that are cited inthe summary or detailed description of the invention are herebyincorporated by reference, in their entirety.

While the invention here has been described in connection with specificembodiments thereof, it will be understood that it is capable of furthermodifications and this application is intended to cover any variations,uses, or adaptions of the invention following, in general, theprinciples of the invention and including such departures from thepresent disclosure as come within known or customary practice within theart to which the invention pertains and as may be applied to theessential features hereinbefore set forth and as follows in the scope ofthe appended claims.

1. A purified polypeptide comprising a portion of a mammalian Flt4receptor tyrosine kinase (Flt4) extracellular domain (EC), said portionconsisting esstentially of the first, second, and third immunoglobulindomains of the Flt4-EC. 2-10. (canceled)
 11. A soluble polypeptideaccording to claim
 1. 12. A polynucleotide comprising a nucleotidesequence that encodes the polypeptide of claim
 1. 13. A polynucleotideaccording to claim 12, further comprising an expression control sequenceoperatively linked to the sequence the encodes the polypeptide.
 14. Anexpression vector comprising an expression control sequence operativelylinked to a polynucleotide according to claim
 12. 15. An expressionvector according to claim 14, wherein the expression control sequencecomprises a promoter that promotes expression in a mammalian cell. 16.An expression vector according to claim 15 that is a viral vectorselected from the group consisting of retrovirus, adenovirus,adeno-associated virus, vaccinia virus and herpesvirus.
 17. A host celltransformed or transfected with a vector according to claim
 15. 18-19.(canceled)